Ionizable cationic lipids

ABSTRACT

The present disclosure provides compounds useful as ionizable cationic lipids. The ionizable cationic lipids are useful for preparing lipid nanoparticles for the delivery of therapeutic nucleic acids to cells. Cationic ionizable lipids were engineered with improved stability to oxidative degradation while in storage.

RELATED APPLICATION

This patent application claims the benefit of and priority to U.S.Provisional Patent Application No. 63/118,534, filed Nov. 25, 2020,incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

This specification includes a sequence listing submitted herewith, whichincludes the file entitled 191016-010402_ST25.txt having the followingsize: 1,270 bytes which was created Nov. 23, 2021, the contents of whichare incorporated by reference herein.

FIELD

The present disclosure relates to the field of medicine. In someembodiments, the present disclosure provides ionizable cationic lipids,uses of ionizable cationic lipids in pharmaceutical compositions such asvaccines, and methods of using these compositions including dendriticcell targeting.

BACKGROUND

Lipid nanoparticles (LNP) are used for the delivery of therapeuticnucleic acids to cells. For example, LNP pharmaceutical compositions areemployed in vaccines to deliver mRNA therapeutics. LNP formulationstypically include an ionizable cationic lipid (ICL). However, it isknown in the art that certain ICL compounds are undesirably sensitive tooxidation during storage. Therefore, there is a need for improved ICLcompounds with improved stability to oxidative degradation while instorage, while also providing desired transfection activity or potencyin cells when incorporated in a LNP with a therapeutic agent such as anucleic acid.

SUMMARY

Stabilized Nucleic Acid Lipid Particles (SNALP) are used as a vehiclefor the systemic delivery of mRNA or other nucleic acid therapeutics.SNALP compositions include cationic lipids such as MC3 or KC2,comprising a protonatable tertiary amine head group joined to a pair oflinear 18 carbon aliphatic chains containing a pair of carbon-carbondouble bonds separated by a single methylene group (e.g., linoleicacid). However, while the structure of these hydrocarbon chains, eachcontaining a pair of double bonds separated by a single methylene group,imparts desirable biological properties to the SNALP compositions, thischemical sub-structure also results in the undesired problem ofincreased sensitivity of the compound to oxidative degradation. Forexample, FIG. 1 is a depiction of the oxidative degradation mechanismsof lipid esters of linoleic acid containing conjugated multipleunsaturations that are particularly sensitive to oxidation. What isneeded are novel cationic lipids suitable for use in a SNALPcomposition, but having enhanced resistance to oxidative degradation.

The present disclosure provides for compositions of ionizable cationiclipids useful in the preparation of lipid nanoparticles (LNP) for thedelivery of therapeutic nucleic acids to cells. Cationic lipids can beengineered with improved stability to oxidative degradation while instorage, while retaining high transfection activity or potency in cells.In some embodiments, lipids disclosed herein comprise at least twocarbon-carbon double bonds (olefins) spaced with at least two methylenegroups. The olefins in the lipid tails separated by at least twomethylene groups render the ionizable cationic lipid compounds describedherein considerably less susceptible to oxidation compared to compoundsseparated by one methylene group, for example DLin-MC3-DMA, consideredthe gold standard in ionizable cationic lipid design and which wasreported to have stability issues.

In some embodiments, the lipids are designed to be biodegradable, thusimproving the tolerability of nanoparticles formed with them in vivo. Insome embodiments, compositions further comprising ligands, such asantibody conjugates, directed against cell surface receptors to targetlipid nanoparticles in a highly specific manner to dendritic cells areprovided.

In some embodiments, the ionizable lipid is a cationic lipid selectedfrom the group consisting of: AKG-UO-1, AKG-UO-2, AKG-UO-4, AKG-UO-4Aand AKG-UO-5. In some embodiments, the ionizable lipid is AKG-UO-1:

In some embodiments, the ionizable lipid is AKG-UO-1A:

In some embodiments, the ionizable lipid is AKG-UO-1B:

In some embodiments, the ionizable lipid is AKG-UO-2

In some embodiments, the ionizable lipid is AKG-UO-4:

In some embodiments, the ionizable lipid is AKG-UO-4A:

In some embodiments, the ionizable lipid is AKG-UO-5:

In some embodiments, the ionizable lipid is AKG-UO-6, AKG-UO-7,AKG-UO-7, AKG-UO-8, AKG-UO-9, or AKG-UO-10:

In some embodiments, the ionizable lipid comprises a head group thatincludes a methylated phosphate moiety. In some embodiments, theionizable lipid is selected from the group consisting of Compounds 20-22and Compounds 26-28:

In some embodiments the ionizable lipid is AKG-UO-3:

In some embodiments, the ionizable lipid is selected from the groupconsisting of Compound 1-8:

In some embodiments, the ionizable lipid is a compound selected from thegroup consisting of Compounds 9-19:

In some embodiments, the ionizable lipid is a compound selected from thegroup consisting of Compounds 29-34:

In some embodiments, the ionizable lipid is selected from the groupconsisting of Compounds 35-38:

In some embodiments, the compounds provided herein have greater than30%, greater 50%, greater 75%, greater 90%, and greater 95% reduction inoxidation byproducts when compared to the control LNP. In someembodiments, the compounds provided herein have greater than 30%,greater 50%, greater 75%, greater 90%, and greater 95% reduction inoxidation byproducts when compared to the control LNP containing theDLin-KC2-DMA lipid.

In some embodiments, the ionizable lipid encapsulate the nucleic acid.In some embodiments, the nucleic acid is a siRNA molecule. In someembodiments, the nucleic acid is a mRNA molecule. In some embodiments,the nucleic acid is a DNA molecule. In some embodiments, the nucleicacid is mRNA. In some embodiments, the nucleic acid is siRNA. In someembodiments, the nucleic acid is DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of the oxidative degradation mechanisms of lipidesters of linoleic acid containing conjugated multiple unsaturationsthat are particularly sensitive to oxidation.

FIG. 2 is a graph showing a comparison of transfection activity for LNPsprepared with KC2 and KC3 polyunsaturated ICLs with a single methylenebetween the two olefins and LNPs prepared with KC2-01 and KC3-01 ICLswith four methylenes between the two olefins.

FIG. 3 is a graph showing the impact of ionizable lipid acyl chaincomposition on transfection efficiency of mCherry mRNA LNPs in dendriticcells.

FIG. 4 is a graph showing the oxidative degradation of lipid suspensionsof ICLs with a single methylene between two olefins (KC2, KC3, andO-11769) and those with four methylenes between the two olefins (KC2-01,KC3-01, and UO-1).

FIG. 5 is a graph showing the oxidative degradation of liposomescontaining O-11769, an ICL with a single methylene between two olefinsliposomes containing UO-1, an ICL with four methylenes between the twoolefins.

FIG. 6 is a schematic of the reaction of the reduced c-terminal cysteineof a Fab′ antibody fragment with a maleimide terminated-poly(ethyleneglycol) 2000 derivatized distearoylphosphatidylethanolamine.

FIG. 7 is a scheme of the synthesis of acid intermediates for AKG-UO-1to AKG-UO-3 (Scheme 1).

FIG. 8 is a scheme of the synthesis of acid intermediates for AKG-BDG-01and AKG-BDG-02 (Scheme 3).

FIG. 9 is a scheme of the synthesis of AKG-UO-2 (Scheme 5).

FIG. 10 is a scheme of the synthesis of AKG-UO-3 (Scheme 6).

FIG. 11A shows Scheme 7; FIG. 11B shows Scheme 8 and FIG. 11C showsScheme 9.

FIG. 12 is a scheme of the synthesis of(S)-4-(dimethylamino)butane-1,2-diyl(6Z,6′Z,11Z,11′Z)-bis(octadeca-6,11-dienoate)(AKG-UO-1a).

FIG. 13 is a scheme of the synthesis of Synthesis of2-((S)-2,2-di((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine(AKG-KC2-01, O-12095) and of3-((S)-2,2-di((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine(AKG-KC3-01, O-12096).

FIG. 14 is a scheme of the synthesis of Synthesis of2-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine(AKG-KC2-OA, O-11880);2-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine(AKG-KC2-PA, O-11879); and3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine(AKG-KC3-OA, O-11957).

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the compositions and methods of the presentdisclosure.

Disclosed herein are compounds, compositions and methods related to thetreatment of bacterial infections. As used herein, the term “compound”,“drug” and “active agent” are used interchangeably. Some aspects of thedisclosure relate to novel ionizable lipids or bioreducible ionizablelipids. These lipids are cationic (i.e. positively charged) at acidicpH, such as encountered intracellularly following endocytosis orphagocytosis by a cell. The same lipids, and compositions containingthem, are near neutral in charge when present at pH 7.4. These lipidsmay also have multiple olefins that are separated by at least twomethylene groups present in their alkyl or acyl groups.

Some aspects of the disclosure relate to the process for the synthesisof the novel ionizable lipids.

Other aspects relate to compositions comprising lipidic nanoparticlescomprising ionizable cationic lipid, the lipidic nanoparticlescontaining nucleic acids. In some embodiments, nucleic acids areencapsulated into the lipidic nanoparticles.

Other aspects of the disclosure relate to the use of these ionizablelipids or lipidic nanoparticles compositions comprising ionizable lipidsin vaccines for the prevention of infectious diseases or cancer. In someembodiments, the infectious disease can be a bacterial or a viralinfection. In some embodiments, the compositions described herein can beused to prevent infections related to tuberculosis, HIV/AIDS, malaria,or coronavirus-related infections such as COVID-19. In otherembodiments, the infection is influenza, hepatitis B, hepatitis C,Dengue, human papillomavirus (HPV), norovirus, mumps, measles,Meningococcal disease, pneumococcal disease, polio, rotovirus,respiratory syncytial virus (RSV), rubella, shingles/herpes zostervirus, tetanus, or whooping cough.

In some embodiments, the compounds and compositions described herein maypromote efficient uptake and transfection of target cells, includingtissue macrophages and dendritic cells. The efficient delivery nucleicacids coding for antigen specific for infectious viruses or bacteria,and subsequent presentation of that antigen to elicit the desired immuneresponse to protect against corresponding infections is a result.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs.

As used herein, the following terms and phrases are intended to have thefollowing meanings:

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that arepresent in a given embodiment, yet open to the inclusion of unspecifiedelements.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the disclosure.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

The term “comprising” when used in the specification includes“consisting of” and “consisting essentially of”.

If it is referred to “as mentioned above” or “mentioned above”, “supra”within the description it is referred to any of the disclosures madewithin the specification in any of the preceding pages.

If it is referred to “as mentioned herein”, “described herein”,“provided herein,” or “as mentioned in the present text,” or “statedherein” within the description it is referred to any of the disclosuresmade within the specification in any of the preceding or subsequentpages.

As used herein, the term “about” means acceptable variations within 20%,within 10% and within 5% of the stated value. In certain embodiments,“about” can mean a variation of +/−1%, 2%, 3%, 4%, 5%, 10% or 20%.

The term “effective amount” as used herein with respect to a compound orthe composition means the amount of active compound (also referredherein as active agent or drug) sufficient to cause a bactericidal orbacteriostatic effect. In one embodiment, the effective amount is a“therapeutically effective amount” meaning the amount of active compoundthat is sufficient alleviate the symptoms of the bacterial infectionbeing treated.

The term “subject” (or, alternatively, “patient”) as used herein refersto an animal, preferably a mammal, most preferably a human that receiveseither prophylactic or therapeutic treatment.

The term “administration” or “administering” as used herein includes allmeans of introducing the compounds or the pharmaceutical compositions tothe subject in need thereof, including but not limited to, oral,intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal,inhalation, buccal, ocular, sublingual, vaginal, rectal and the like.Administration of the compound or the composition is suitablyparenteral. For example, the compounds or the composition can bepreferentially administered intravenously, but can also be administeredintraperitoneally or via inhalation like is currently used in the clinicfor liposomal amikacin in the treatment of Mycobacterium avium (seeShirley et al., Amikacin Liposome Inhalation Suspension: A Review inMycobacterium avium Complex Lung Disease. Drugs. 2019 April;79(5):555-562)

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic or preventative measures such as those described herein.

The term “pharmaceutically acceptable salt” refers to a relativelynon-toxic, inorganic or organic acid addition salt of a compound of thepresent disclosure which salt possesses the desired pharmacologicalactivity.

The term “alkyl” means saturated carbon chains having from one to twentycarbon atoms which may be linear or branched or combinations thereof,unless the carbon chain is defined otherwise. Examples of alkyl groupsinclude methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl,pentyl, hexyl, heptyl, octyl, and the like. Unless stated otherwisespecifically in the specification, an alkyl group is optionallysubstituted.

The term “lipidic nanoparticle”, or “LNP”, refers to particles having adiameter of from about 5 to 500 nm. In some embodiments, the lipidnanoparticle comprises one or more active agents. In some embodiments,the lipid nanoparticle comprises a nucleic acid. In some embodiments,the nucleic acid is condensed in the interior of the nanoparticle with acationic lipid, polymer, or polyvalent small molecule and an externallipid coat that interacts with the biological milieu. Due to therepulsive forces between phosphate groups, nucleic acids are naturallystiff polymers and prefer elongated configurations. In the cell, to copewith volume constraints DNA can pack itself in the appropriate solutionconditions with the help of ions and other molecules.

Usually, DNA condensation is defined as the collapse of extended DNAchains into compact, orderly particles containing only one or a fewmolecules. By binding to phosphate groups, cationic lipidic can condenseDNA by neutralizing the phosphate charges and allow close packing.

In some embodiments, the active agent is encapsulated into the LNP. Insome embodiments, the active agent can be an anionic compounds, forexample, but not limited to DNA, RNA, natural and syntheticoligonucleotides (including antisense oligonucleotides, interfering RNAand small interfering RNA), nucleoprotein, peptide, nucleic acid,ribozyme, DNA-containing nucleoprotein, such as an intact or partiallydeproteinated viral particles (virions), oligomeric and polymericanionic compounds other than DNA (for example, acid polysaccharides andglycoproteins)). In some embodiments, the active agent can be intermixedwith an adjuvant.

In a LNP vaccine product, the active agent is generally contained in theinterior of the LNP. In some embodiments, the active agent comprises anucleic acid. Typically, water soluble nucleic acids are condensed withcationic lipids or polycationic polymers in the interior of the particleand the surface of the particle is enriched in neutral lipids orPEG-lipid derivatives. Additional ionizable cationic lipid may also beat the surface and respond to acidification in the environment bybecoming positively charged, facilitating endosomal escape.

Ionizable lipids can have different properties or functions with respectto LNPs. Due to the pKa of the amino group, the lipid molecules canbecome positively charged in acidic conditions.

Under these conditions, lipid molecules can electrostatically bind tothe phosphate groups of the nucleic acid which allows the formation ofLNPs and the entrapment of the nucleic acid. In some embodiments, thepKa can be low enough that it renders the LNP substantially neutral insurface charge in biological fluids, such as blood, which are atphysiological pH values. High LNP surface charge is associated withtoxicity, rapid clearance from the circulation by the fixed and freemacrophages, hemolytic toxicities, including immune activation (Filionet al Biochim Biophys Acta. 1997 Oct. 23; 1329(2):345-56).

In some embodiments, pKa can be high enough that the ionizable cationiclipid can adopt a positively charged form at acidic endosomal pH values.This way, the cationic lipids can combine with endogenous endosomalanionic lipids to promote membrane lytic nonbilayer structures such asthe hexagonal HII phase, resulting in more efficient intracellulardelivery. In some embodiments, the pKa ranges between 6.2-6.5. Forexample, the pKa can be about 6.2, about 6.3, about 6.4, about 6.5.Unsaturated tails also contribute to the lipids' ability to adoptnonbilayer structures. (Jayaraman et al., Angew Chem Int Ed Engl. 2012Aug. 20; 51(34):8529-33).

Release of nucleic acids from LNP formulations, among othercharacteristics such as liposomal clearance and circulation half-life,can be modified by the presence of polyethylene glycol and/or sterols(e.g. cholesterol) or other potential additives in the LNP, as well asthe overall chemical structure, including pKa of any ionizable cationiclipid included as part of the formulation.

The term “bioreducible” refers to compounds that undergo accelerateddegradation due to the cleavage of disulfide linkages in a reductiveenvironment. Unlike other nucleic acid therapeutics such as siRNA, thesuccess of mRNA-based therapies depends on the availability of a safeand efficient delivery vehicle that encapsulates the mRNA. mRNA isfragile and needs a protective coating for it to remain active until itreaches its target site. mRNA containing LNPs are a promising vaccineoption for Covid-19 immunity (Jackson et al., Preliminary Report. N EnglJ Med. 2020 Nov. 12; 383(20):1920-1931). The efficiency and tolerabilityof LNPs has been attributed to the amino lipid and unlike manybiomaterial applications that may have a required service lifetime ofweeks or months, functional LNP mediated delivery of mRNA occurs withinhours obviating the need for persistent lipids. Indeed in applicationswhere chronic dosing is required this will be especially important. Ithas been demonstrated that LNPs enter cells via endocytosis andaccumulate in endolysosomal compartments. ICL is able to effectivelydeliver mRNA to the cytosol after endocytosis while being susceptible toenzymatic hydrolysis in late endosomes/lysosomes by lipases orhydrolysis triggered by the reductive environment of the lysosomeallowing complete biodegradation. The extracellular space is arelatively oxidative environment, while the intracellular space is areductive one, allowing a disulfide linked molecule to remain intact inthe extracellular space but be rapidly reduced once internalized (Huanget al., Mol Ther. 2005 March; 11(3):409-17, 2005). Some embodiments,provide bioreducible disulfide linked ICL molecules (see compounds29-36, Table 2) that are stable in LNP formulation and while incirculation but undergo cleavage in the reductive environment of thelysosome. Such compounds and compositions can facilitate rapidbiological destruction of the lipids and can prevent potentially toxicaccumulation of ICL lipids (as observed in rats with DLin-MC3-DMA(Sabins et al., Mol Ther. 2018 Jun. 6; 26(6):1509-1519).

The terms “encapsulation” and “entrapped,” as used herein, refer to theincorporation or association of the mRNA, DNA, siRNA or other nucleicacid pharmaceutical agent in or with a lipidic nanoparticle. As usedherein, the term “encapsulated” refers to complete encapsulation orpartial encapsulation. A siRNA may be capable of selectively knockingdown or down regulating expression of a gene of interest. For example,an siRNA could be selected to silence a gene associated with aparticular disease, disorder, or condition upon administration to asubject in need thereof of a nanoparticle composition including thesiRNA. A siRNA may comprise a sequence that is complementary to an mRNAsequence that encodes a gene or protein of interest.

The term “mol %” with regard to cholesterol refers to the molar amountof cholesterol relative to the sum of the molar amounts of cholesteroland non-PEGylated phospholipid expressed in percentage points. Forexample, “55 mol. % cholesterol” in a liposome containing cholesteroland HSPC refers to the composition of 55 mol. parts of cholesterol per45 mol. parts of HSPC.

The term “mol %” with regard to PEG-lipid refers to the ratio of themolar amount of PEG-lipid and non-PEGylated phospholipid expressed inpercentage points. For example, “5 mol. % PEG-DSPE” in a LNP containingHSPC and PEG-DSPE refers to the composition having 5 mol. parts ofPEG-DSPE per 100 mol. parts of HSPC.

As used herein, the term “pharmaceutically acceptable carrier, diluentor excipient” includes without limitation any adjuvant, carrier,excipient, glidant, sweetening agent, diluent, preservative,dye/colorant, flavor enhancer, surfactant, wetting agent, dispersingagent, suspending agent, stabilizer, isotonic agent, solvent, oremulsifier which has been approved by the United States Food and DrugAdministration as being acceptable for use in humans or domesticanimals.

Various aspects and embodiments are described in further detail in thefollowing subsections.

Compounds

Disclosed herein are compounds of Formula I, Formula II, Formula III,Formula IV or pharmaceutically acceptable salts thereof that are usefulin the preparation of vaccines. Also disclosed herein are compositionscomprising the cationic lipids of Formula I, Formula II, Formula III,Formula IV or pharmaceutically acceptable salts thereof. In someembodiments, the vaccine is used for the prevention Mycobacteriuminfections. In some embodiments, the vaccine can be used for theprevention of tuberculosis, nontuberculous mycobacteria (NTM),nontuberculosis lung disease, leprosy, Mycobacteriumavium-intracellulare, Mycobacterium kansasii, Mycobacterium marinum,Mycobacterium ulcerans, Mycobacterium chelonae, Mycobacterium fortuitum,Mycobacterium abscessus and other infectious diseases such ascoronaviruses (COVID-19, SARS CoV2, SARS-CoV, MERS-CoV), diphtheria,ebola, flu (Influenza), hepatitis, Hib disease, HIV/AIDS, HPV (HumanPapillomavirus), malaria, measles, meningococcal disease, mumps,norovirus, plague, pneumococcal disease, polio, respiratory syncytialvirus (RSV), rotavirus, rubella (German Measles), shingles (HerpesZoster), tetanus (Lockjaw), whooping cough (Pertussis) and zika.

Provided herein are compounds, compositions and methods for thetreatment or prevention of infectious diseases, including tuberculosis.According to aspects of the disclosure, the cationic lipids comprise thecompounds having Formula I, II, III or IV or pharmaceutically acceptablesalts thereof. In some embodiments, the cationic lipids two fatty acylgroups as in Formula II, II, III or IV. Lipids disclosed herein compriseat least two carbon-carbon double bonds (olefins) spaced with at leasttwo methylene or substituted methylene groups, wherein the substitutedmethylene is —C(R₁)(R₂)— wherein R₁ and R₂ are independently H, alkyl,or halogen.

One aspect of the disclosure provides a compound of Formula I orpharmaceutically acceptable salts thereof:

wherein Y is independently a methyl or ethyl group,wherein the two fatty acyl groups have between 16-18 carbons and containtwo unconjugated olefins.

In some embodiments, the two fatty acyl groups have 16 carbons. In someembodiments, the two fatty acyl groups have 17 carbons. In someembodiments, the two fatty acyl groups have 18 carbons.

Another aspect of the disclosure provides for a compound of Formula IIor pharmaceutically acceptable salts thereof:

wherein R is a substituent comprising a dialkylamino group of one of thestructures shown above, wherein the two fatty acyl groups are between16-18 carbons and contain two olefins that are separated by at least twomethylene groups.

In some embodiments, the two fatty acyl groups have 16 carbons. In someembodiments, the two fatty acyl groups have 17 carbons. In someembodiments, the two fatty acyl groups have 18 carbons.

Another aspect of the disclosure provides for a compound of Formula IIIor pharmaceutically acceptable salts thereof:

wherein Y is a methyl or ethyl group,wherein the two fatty acyl groups are disulfide fatty acyl groups havingbetween 16-18 carbons and containing a single olefin.

In some embodiments, the two fatty acyl groups have 16 carbons. In someembodiments, the two fatty acyl groups have 17 carbons. In someembodiments, the two fatty acyl groups have 18 carbons.

In some embodiments, the compound in Formula I-III has a pKa between 6and 7.

In some embodiments, a lipidic nanoparticle composition comprises lipidsand nucleic acids, the lipidic nanoparticles comprising a compound ofFormula I, II, III, combinations thereof or pharmaceutically acceptablesalts thereof.

In some embodiments, an LNP comprises an ionizable lipid having astructure of Formula (IV):

or a pharmaceutically acceptable salt thereof,wherein Y is

each R²² is independently alkyl, alkenyl, alkynyl, or heteroalkyl, eachof which is optionally substituted with R^(B); each R^(B) isindependently alkyl, halo, hydroxy, amino, cycloalkyl, or heterocyclyl;n is an integer between 1 and 10 (inclusive); and

denotes the attachment point.

In some embodiments, Y is.

In some embodiments, the compound in Formula IV has a pKa between 6 and7.

In some embodiments, the compounds have the structure of the compoundslisted in Table 1 or Table 2. Table 1 shows examples of cationic lipids.Table 2 shows examples of bioreducible cationic lipids.

In some embodiments, the compound has a structure as shown in Table 1.

TABLE 1 Exemplary cationic lipids

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

In some embodiments, the compound has a structure as shown in Table 2.In some embodiments, the compound has a structure as shown in Table 2and is bioreducible.

TABLE 2 Exemplary bioreducible cationic lipids

29

30

31

32

33

34

35

36

37

38

The present disclosure features a lipid nanoparticle comprising nucleicacids such as DNA, mRNA, siRNA, antisense oligonucleotides, CRISPRcomponents such as a guide RNA (gRNA or sgRNA) and a CRISPR-associatedendonuclease (Cas protein) and a lipid. Exemplary lipids includeionizable cationic lipids (ICLs), phospholipids, sterol lipids, alkyleneglycol lipids (e.g., polyethylene glycol lipids), sphingolipids,glycerolipids, glycerophospholipids, prenol lipids, saccharolipids,fatty acids, and polyketides. In some embodiments, the LNP comprises asingle type of lipid. In some embodiments, the LNP comprises a plurality(e.g. two or more) of lipids. An LNP may comprise one or more of anionizable cationic lipid, a phospholipid, a sterol, or an alkyleneglycol lipid (e.g., a polyethylene glycol lipid).

In an embodiment, the LNP comprises an ionizable cationic lipid. As usedherein “ionizable cationic lipid”, “ionizable lipid” and “ICL” are usedinterchangeably. An ICL is a lipid that comprises an ionizable moietycapable of bearing a charge (e.g., a positive charge e.g., a cationiclipid) under certain conditions (e.g., at a certain pH range, e.g.,under physiological conditions). The ionizable moiety may comprise anamine, and preferably a substituted amine. An ionizable lipid may be acationic lipid or an anionic lipid. In addition to an ionizable moiety,an ionizable lipid may contain an alkyl or alkenyl group, e.g., greaterthan six carbon atoms in length (e.g., greater than about 8 carbons, 10carbons, 12 carbons, 14 carbons, 16 carbons, 18 carbons, 20 carbons ormore in length). Additional ionizable lipids that may be included in anLNP described herein are disclosed in Jayaraman et al. (Angew. Chem.Int. Ed. 51:8529-8533 (2012)), Semple et al. Nature Biotechnol.28:172-176 (2010)), and U.S. Pat. Nos. 8,710,200 and 8,754,062, each ofwhich is incorporated herein by reference in its entirety.

In some embodiments, an LNP comprises an ionizable lipid having astructure of Formula (IV).

or a pharmaceutically acceptable salt thereof,wherein Y is

each R²² is independently alkyl, alkenyl, alkynyl, or heteroalkyl, eachof which is optionally substituted with R^(B); each R^(B) isindependently alkyl, halo, hydroxy, amino, cycloalkyl, or heterocyclyl;n is an integer between 1 and 10 (inclusive); and

denotes the attachment point.

In some embodiments, Y is

An LNP may comprise an ionizable lipid at a concentration greater thanabout 0.1 mol %, e.g., of the total lipid composition of the LNP. In anembodiment, the LNP comprises an ionizable lipid at a concentration ofgreater than about 1 mol %, about 2 mol %, about 4 mol %, about 8 mol %,about 20 mol %, about 40 mol %, about 50 mol %, about 60 mol %, about 80mol %, e.g., of the total lipid composition of the LNP. In anembodiment, the LNP comprises an ionizable lipid at a concentration ofgreater than about 20 mol %, about 40 mol %, or about 50 mol %. In anembodiment, the LNP comprises an ionizable lipid at a concentrationbetween about 1 mol % to about 95 mol %, e.g., of the total lipidcomposition of the LNP. In an embodiment, the LNP comprises an ionizablelipid at a concentration between about 2 mol % to about 90 mol %, about4 mol % to about 80 mol %, about 10 mol % to about 70 mol %, about 20mol % to about 60 mol %, about 40 mol % to about 55 mol %, e.g., of thetotal lipid composition of the LNP. In an embodiment, the LNP comprisesan ionizable lipid at a concentration between about 20 mol % to about 60mol %. In an embodiment, the LNP comprises an ionizable lipid at aconcentration between about 40 mol % to about 55 mol %.

In an embodiment, the LNP comprises a phospholipid. A phospholipid is alipid that comprises a phosphate group and at least one alkyl, alkenyl,or heteroalkyl chain. A phospholipid may be naturally occurring ornon-naturally occurring (e.g., a synthetic phospholipid). A phospholipidmay comprise an amine, amide, ester, carboxyl, choline, hydroxyl,acetal, ether, carbohydrate, sterol, or a glycerol. In some embodiments,a phospholipid may comprise a phosphocholine, phosphosphingolipid, or aplasmalogen. Exemplary phospholipids include1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soyphosphatidylcholine (HSPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine(DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC),1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DAPC),1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC),1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC),1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC),1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),bis(monoacylglycerol)phosphate (BMP), L-α-phosphatidylcholine,1,2-Diheptadecanoyl-sn-glycero-3-phosphorylcholine (DHDPC), and1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC). Additionalphospholipids that may be included in an LNP described herein aredisclosed in Li, J. et al. (Asian J. Pharm. Sci. 10:81-98 (2015)), whichis incorporated herein by reference in its entirety.

In some embodiments, an LNP comprises a phospholipid having a structureof Formula (V):

or a pharmaceutically acceptable salt thereof, wherein each R²³ isindependently alkyl, alkenyl, or heteroalkyl; wherein each alkyl,alkenyl, or heteroalkyl is optionally substituted with R^(C); each R²⁵is independently hydrogen or alkyl; R²⁴ is absent, hydrogen, or alkyl;each R^(C) is independently alkyl, halo, hydroxy, amino, cycloalkyl, orheterocyclyl; m is an integer between 1 and 4 (inclusive); and u is 2 or3.

In some embodiments, each R²³ is independently alkyl (e.g., C₂-C₃₂alkyl, C₄-C₂₈ alkyl, C₈-C₂₄ alkyl, C₁₂-C₂₂ alkyl, or C₁₆-C₂₀ alkyl). Insome embodiments, each R²³ is independently alkenyl (e.g., C₂-C₃₂ alkyl,C₄-C₂₈ alkenyl, C₈-C₂₄ alkenyl, C₁₂-C₂₂ alkenyl, or C₁₆-C₂₀ alkenyl). Insome embodiments, each R²³ is independently heteroalkyl (e.g.,C₄-C₂₈heteroalkyl, C₈-C₂₄heteroalkyl, C₁₂-C₂₂heteroalkyl,C₁₆-C₂₀heteroalkyl). In some embodiments, each R²³ is independentlyC₁₆-C₂₀ alkyl. In some embodiments, each R²³ is independently C₁₇ alkyl.In some embodiments, each R²³ is independently heptadecyl. In someembodiments, each R²³ is the same. In some embodiments, each R²³ isdifferent. In some embodiments, each R²³ is optionally substituted withR^(C). In some embodiments, R^(C) is independently alkyl, halo, hydroxy,amino, cycloalkyl, or heterocyclyl.

In some embodiments, one of R²⁵ is hydrogen. In some embodiments, one ofR²⁵ is alkyl. In some embodiments, one of R²⁵ is methyl. In someembodiments, each R²⁵ is independently alkyl. In some embodiments, eachR²⁵ is independently methyl. In some embodiments, each R²⁵ isindependently methyl and u is 2. In some embodiments, each R²⁵ isindependently methyl and u is 3.

In some embodiments, R²⁴ is absent, and the oxygen to which it isattached carries a negative charge. In some embodiments, R²⁴ ishydrogen.

In some embodiments, m is an integer between 1 and 10, 1 and 8, 1 and 6,1 and 4. In some embodiments, m is 1, 2, 3, or 4. In some embodiments, mis 1. In some embodiments, m is 2. In some embodiments, m is 3. In someembodiments, the phospholipid is1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments,the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Insome embodiments, the phospholipid is1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In some embodiments,the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE).

An LNP may comprise a phospholipid at a concentration greater than about0.1 mol %, e.g., of the total lipid composition of the LNP. In anembodiment, the LNP comprises a phospholipid at a concentration ofgreater than about 0.5 mol %, about 1 mol %, about 1.5 mol %, about 2mol %, about 3 mol %, about 4 mol %, about 5 mol %, about 6 mol %, about8 mol %, about 10 mol %, about 12 mol %, about 15 mol %, about 20 mol %,about 50 mol %, e.g., of the total lipid composition of the LNP. In anembodiment, the LNP comprises a phospholipid at a concentration ofgreater than about 1 mol %, about 5 mol %, or about 10 mol %. In anembodiment, the LNP comprises a phospholipid at a concentration betweenabout 0.1 mol % to about 50 mol %, e.g., of the total lipid compositionof the LNP. In an embodiment, the LNP comprises a phospholipid at aconcentration between about 0.5 mol % to about 40 mol %, about 1 mol %to about 30 mol %, about 5 mol % to about 25 mol %, about 10 mol % toabout 20 mol %, about 10 mol % to about 15 mol %, or about 15 mol % toabout 20 mol %, e.g., of the total lipid composition of the LNP. In anembodiment, the LNP comprises a phospholipid at a concentration betweenabout 5 mol % to about 25 mol %. In an embodiment, the LNP comprises aphospholipid at a concentration between about 10 mol % to 20 mol %.

In an embodiment, the LNP comprises a sterol or ionizable sterolmolecule. A sterol is a lipid that comprises a polycyclic structure andan optionally a hydroxyl or ether substituent, and may be naturallyoccurring or non-naturally occurring (e.g., a synthetic sterol). Sterolsmay comprise no double bonds, a single double bond, or multiple doublebonds. Sterols may further comprise an alkyl, alkenyl, halo, ester,ketone, hydroxyl, amine, polyether, carbohydrate, or cyclic moiety.Sterol may further contain a bioreducible disulfide linkage between thedialkylamino group and the polycyclic portion of the molecule (see Table2, Compounds 35-38). An exemplary listing of sterols includescholesterol, dehydroergosterol, ergosterol, campesterol, β-sitosterol,stigmasterol, lanosterol, dihydrolanosterol, desmosterol,brassicasterol, lathosterol, zymosterol, 7-dehydrodesmosterol,avenasterol, campestanol, lupeol, and cycloartenol. In some embodiments,the sterol comprises cholesterol, dehydroergosterol, ergosterol,campesterol, β-sitosterol, or stigmasterol. Additional sterols that maybe included in an LNP described herein are disclosed in Fahy, E. et al.(J. Lipid. Res. 46:839-862 (2005).

Ionizable Sterols

In some embodiments, an LNP comprises a sterol having a structure ofFormula (VI): (VI) or a pharmaceutically acceptable salt thereof,wherein R²⁶ is hydrogen, alkyl, heteroalkyl, or —C(O)R^(D), R²⁷ ishydrogen, alkyl, or —OR^(E); each of R^(D) and R^(E) is independentlyhydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl orheteroaryl, wherein each alkyl, alkenyl, heteroalkyl, cycloalkyl,heterocyclyl, aryl or heteroarylis optionally substituted with alkyl,halo, or carbonyl; and each “ ” is either a single or double bond, andwherein each carbon atom participating in the single or double bond isbound to 0, 1, or 2 hydrogens, valency permitting.

In some embodiments, one of “ ” is a single bond. In some embodiments,one of “ ” is a double bond. In some embodiments, two of a “ ” aresingle bonds. In some embodiments, two of “

” are double bonds. In some embodiments, each “

” is a single bond. In some embodiments, each “

” is a double bond.

In some embodiments, the sterol is cholesterol. In some embodiments, thesterol is dehydroergosterol. In some embodiments, the sterol isergosterol. In some embodiments, the sterol is campesterol. In someembodiments, the sterol is β-sitosterol. In some embodiments, the sterolis stigmasterol. In some embodiments, the sterol is a corticosteroid.(e.g., corticosterone, hydrocortisone, cortisone, or aldosterone) An LNPmay comprise a sterol at a concentration greater than about 0. mol %,e.g., of the total lipid composition of the LNP. In an embodiment, theLNP comprises a sterol at a concentration greater than about 0.5 mol %,about 1 mol %, about 5 mol %, about 10 mol %, about 15 mol %, about 20mol %, about 25 mol %, about 35 mol %, about 40 mol %, about 45 mol %,about 50 mol %, about 55 mol %, about 60 mol %, about 65 mol %, or about70 mol %, e.g., of the total lipid composition of the LNP. In anembodiment, the LNP comprises a sterol at a concentration greater thanabout 10 mol %, about 15 mol %, about 20 mol %, or about 25 mol %. In anembodiment, the LNP comprises a sterol at a concentration between about1 mol % to about 95 mol %, e.g., of the total lipid composition of theLNP. In an embodiment, the LNP comprises a sterol at a concentrationbetween about 5 mol % to about 90 mol %, about 10 mol % to about 85 mol%, about 20 mol % to about 80 mol %, about 20 mol % to about 60 mol %,about 20 mol % to about 50 mol %, or about 20 mol % to 40 mol %, e.g.,of the total lipid composition of the LNP. In an embodiment, the LNPcomprises a sterol at a concentration between about 20 mol % to about 50mol %. In an embodiment, the LNP comprises a sterol at a concentrationbetween about 30 mol % to about 60 mol %.

In some embodiments, the LNP comprises an alkylene glycol-containinglipid. An alkylene glycol-containing lipid is a lipid that comprises atleast one alkylene glycol moiety, for example, a methylene glycol or anethylene glycol moiety. In some embodiments, the alkyleneglycol-containing lipid comprises a polyethylene glycol (PEG). Analkylene glycol-containing lipid may be a PEG-containing lipid.Polymer-conjugated lipids may include poly(ethylene glycol)-conjugated(pegylated)phospholipids (PEG-lipids) such as PEG(Mol. weight 2,000)methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glycerol (PEG-DSG),PEG(Mol. weight 2,000) methoxy-poly(ethyleneglycol)-1,2-palmitoyl-sn-glycerol (PEG-DPG), PEG(Mol. weight 2,000)1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000](PEG-DSPE) orN-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethyleneglycol)2000]}(PEG-ceramide). The molecular weight of the PEG portion inthe PEG-lipid component can also vary from 500-10,000 g/mol, from1,500-6000 g/mol, but is preferably about 2,000 MW. Other polymers usedfor conjugation to lipid anchors may include poly(2-methyl-2-oxazoline)(PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), poly-N-vinylpyrrolidone (PVP),polyglycerol, poly(hydroxyethyl L-asparagine) (PHEA), andpoly(hydroxyethyl L-glutamine) (PHEG).

A PEG-containing lipid may further comprise an amine, amide, ester,carboxyl, phosphate, choline, hydroxyl, acetal, ether, heterocycle, orcarbohydrate. PEG-containing lipids may comprise at least one alkyl oralkenyl group, e.g., greater than six carbon atoms in length (e.g.,greater than about 8 carbons, 10 carbons, 12 carbons, 14 carbons, 16carbons, 18 carbons, 20 carbons or more in length), e.g., in addition toa PEG moiety. In an embodiment, a PEG-containing lipid comprises a PEGmoiety comprising at least 20 PEG monomers, e.g., at least 30 PEGmonomers, 40 PEG monomers, 45 PEG monomers, 50 PEG monomers, 100 PEGmonomers, 200 PEG monomers, 300 PEG monomers, 500 PEG monomers, 1000 PEGmonomers, or 2000 PEG monomers. Exemplary PEG-containing lipids includePEG-DMG (e.g., DMG-PEG2k), PEG-c-DMG, PEG-DSG, PEG-DPG, PEG-DSPE,PEG-DMPE, PEG-DPPE, PEG-DOPE, and PEG-DLPE. In some embodiments, thePEG-lipids include PEG-DMG (e.g., DMG-PEG2k), PEG-c-DMG, PEG-DSG, andPEG-DPG. Additional PEG-lipids that may be included in an LNP describedherein are disclosed in Fahy, E. et al. (J. Lipid. Res. 46:839-862(2005) which is incorporated herein by reference in its entirety.

In some embodiments, an LNP comprises an alkylene glycol-containinglipid having a structure of Formula (VII):

or a pharmaceutically acceptable salt thereof, wherein each R²⁸ isindependently alkyl, alkenyl, or heteroalkyl, each of which isoptionally substituted with R^(F); A is absent, O, CH₂, C(O), or NH; Eis absent, alkyl, or heteroalkyl, wherein alkyl or heteroalkyl isoptionally substituted with carbonyl; each R^(F) is independently alkyl,halo, hydroxy, amino, cycloalkyl, or heterocyclyl; and z is an integerbetween 10 and 200 (inclusive).

In some embodiments, each R²⁸ is independently alkyl. In someembodiments, each R²⁸ is independently heteroalkyl. In some embodiments,each R²⁸ is independently alkenyl.

In some embodiments, A is O or NH. In some embodiments, A is CH₂. Insome embodiments, A is carbonyl. In some embodiments, A is absent.

In some embodiments, E is alkyl. In some embodiments, E is heteroalkyl.In some embodiments, both A and E are not absent. In some embodiments, Ais absent. In some embodiments, E is absent. In some embodiments, eitherone of A or E is absent. In some embodiments, both A and E areindependently absent.

In some embodiments, z is an integer between 10 and 200 (e.g., between20 and 180, between 20 and 160, between 20 and 120, between 20 and 100,between 40 and 80, between 40 and 60, between 40 and 50). In someembodiments, z is 45.

In some embodiments, the PEG-lipid is PEG-DMG (e.g., DMG-PEG2k). In someembodiments, the PEG-lipid is α-(3′-{[1,2-di(myristyloxy)propanoxy]carbonylamino}propyl)-ω-methoxy, polyoxyethylene (PEG-c-DMG). In someembodiments, the PEG-lipid is PEG-DSG. In some embodiments, thePEG-lipid is PEG-DPG. An LNP may comprise an alkylene glycol-containinglipid at a concentration greater than about 0.1 mol %, e.g., of thetotal lipid composition of the LNP. In an embodiment, the LNP comprisesan alkylene glycol-containing lipid at a concentration of greater thanabout 0.5 mol %, about 1 mol %, about 1.5 mol %, about 2 mol %, about 3mol %, about 4 mol %, about 5 mol %, about 6 mol %, about 8 mol %, about10 mol %, about 12 mol %, about 15 mol %, about 20 mol %, about 50 mol%, e.g., of the total lipid composition of the LNP. In an embodiment,the LNP comprises an alkylene glycol-containing lipid at a concentrationof greater than about 1 mol %, about 4 mol %, or about 6 mol %. In anembodiment, the LNP comprises an alkylene glycol-containing lipid at aconcentration between about 0.1 mol % to about 50 mol %, e.g., of thetotal lipid composition of the LNP. In an embodiment, the LNP comprisesan alkylene glycol-containing lipid at a concentration between about 0.5mol % to about 40 mol %, about 1 mol % to about 35 mol %, about 1.5 mol% to about 30 mol %, about 2 mol % to about 25 mol %, about 2.5 mol % toabout 20%, about 3 mol % to about 15 mol %, about 3.5 mol % to about 10mol %, or about 4 mol % to 9 mol %, e.g., of the total lipid compositionof the LNP. In an embodiment, the LNP comprises an alkyleneglycol-containing lipid at a concentration between about 3.5 mol % toabout 10 mol %. In an embodiment, the LNP comprises an alkyleneglycol-containing lipid at a concentration between about 4 mol % to 9mol %.

In some embodiments, the LNP comprises at least two types of lipids. Inan embodiment, the LNP comprises two of an ionizable lipid, aphospholipid, a sterol, and an alkylene glycol-containing lipid. In someembodiments, the LNP comprises at least three types of lipids. In anembodiment, the LNP comprises three of an ionizable lipid, aphospholipid, a sterol, and an alkylene glycol-containing lipid. In someembodiments, the LNP comprises at least four types of lipids. In anembodiment, the LNP comprises each of an ionizable lipid, aphospholipid, a sterol, and an alkylene glycol-containing lipid.

The LNP (e.g., as described herein) may comprise one or more of thefollowing components: (i) an ionizable cationic lipid at a concentrationbetween about 1 mol % to about 95 mol % (e.g. about 20 mol % to about 80mol %); (ii) a phospholipid at a concentration between 0.1 mol % toabout 50 mol % (e.g. between about 2.5 mol % to about 20 mol %); (iii) asterol at a concentration between about 1 mol % to about 95 mol % (e.g.about 20 mol % to about 80 mol %); and (iv) a PEG-containing lipid at aconcentration between about 0.1 mol % to about 50 mol % (e.g. betweenabout 2.5 mol % to about 20 mol %). In an embodiment, the LNP comprisesone of (i)-(iv). In an embodiment, the LNP comprises two of (i)-(iv). Inan embodiment, the LNP comprises three of (i)-(iv). In an embodiment,the LNP comprises each of (i)-(iv). In some embodiments, the LNPcomprises (i) and (ii). In some embodiments, the LNP comprises (i) and(iii). In some embodiments, the LNP comprises (i) and (iv). In someembodiments, the LNP comprises (ii) and (iii). In some embodiments, theLNP comprises (ii) and (iv). In some embodiments, the LNP comprises(iii) and (iv). In some embodiments, the LNP comprises (i), (ii), and(iii). In some embodiments, the LNP comprises (i), (ii), and (iv). Insome embodiments, the LNP comprises (ii), (iii), and (iv).

The LNP (e.g., as described herein) may comprise one or more of thefollowing components: (i) Ionizable cationic lipid (ICL) at aconcentration between about 1 mol % to about 95 mol % (e.g. about 20 mol% to about 80 mol %); (ii) DSPC at a concentration between 0.1 mol % toabout 50 mol % (e.g. between about 2.5 mol % to about 20 mol %); (iii)cholesterol at a concentration between about 1 mol % to about 95 mol %(e.g. about 20 mol % to about 80 mol %); and (iv) DMG-PEG2k at aconcentration between about 0.1 mol % to about 50 mol % (e.g. betweenabout 2.5 mol % to about 20 mol %). In an embodiment, the LNP comprisestwo of (i)-(iv). In an embodiment, the LNP comprises three of (i)-(iv).In an embodiment, the LNP comprises each of (i)-(iv). In someembodiments, the LNP comprises (i) and (ii). In some embodiments, theLNP comprises (i) and (iii). In some embodiments, the LNP comprises (i)and (iv). In some embodiments, the LNP comprises (ii) and (iii). In someembodiments, the LNP comprises (ii) and (iv). In some embodiments, theLNP comprises (iii) and (iv). In some embodiments, the LNP comprises(iii) and (iv). In some embodiments, the LNP comprises (i), (ii), and(iii). In some embodiments, the LNP comprises (i), (ii), and (iv). Insome embodiments, the LNP comprises (ii), (iii), and (iv).

In an embodiment, the LNP comprises a ratio of ionizable lipid tophospholipid of about 50:1 to about 1:1 (e.g., 40:1, 32:3, 6:1, 7:1,5:1, 24:5, 26:5, 10:3, 15:2, 16:7, 18:1, 3:1, 3:2, or 1:1). In anembodiment, the LNP comprises a ratio of ionizable lipid to phospholipidof about 15:2. In an embodiment, the LNP comprises a ratio of ionizablelipid to phospholipid of about 5:1. In an embodiment, the LNP comprisesa ratio of ionizable lipid to a sterol of about 10:1 to about 1:10(e.g., 9:1, 8:1, 8:7, 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3:1,2:1, 1:1, 1:2, 1:3, 3:4, 1:4, 3:5, 1:5, 4:5, 1:6, 5:6, 7:6, 7:8, or8:9). In an embodiment, the LNP comprises a ratio of ionizable lipid toan alkylene-containing lipid of about 1:10 to about 10:1 (e.g., 1:9,1:8, 7:8, 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3:1, 2:1, 1:1,1:2, 1:3, 3:4, 1:4, 3:5, 1:5, 4:5, 1:6, 5:6, 7:6, 7:8, or 8:9). In anembodiment, the LNP comprises a ratio of phospholipid to analkylene-containing lipid of about 10:1 to about 1:10 (e.g., 9:1, 8:1,8:7, 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3:1, 2:1, 1:1, 1:2,1:3, 3:4, 1:4, 3:5, 1:5, 4:5, 1:6, 5:6, 7:6, 7:8, or 8:9). In anembodiment, the LNP comprises a ratio of a sterol to analkylene-containing lipid of about 50:1 to about 1:1 (e.g., 40:1, 32:3,6:1, 7:1, 5:1, 24:1, 22:1, 20:1, 22:5, 24:5, 26:5, 10:3, 15:2, 16:7,18:1, 3:1, 3:2, or 1:1).

In an embodiment, a LNP (e.g., described herein) comprises two of anionizable lipid, a phospholipid, a sterol, and an alkyleneglycol-containing lipid (e.g., PEG-containing lipid). In anotherembodiment, a LNP (e.g., described herein) comprises three of anionizable lipid, a phospholipid, a sterol, and an alkyleneglycol-containing lipid (e.g., PEG-containing lipid). In an embodimentLNP (e.g., described herein) comprises each of an ionizable lipid, aphospholipid, a sterol, and an alkylene glycol-containing lipid (e.g.,PEG-containing lipid).

In some embodiments, an LNP described herein has a diameter between 5and 500 nm, e.g., between 10 and 400 nm, 20 and 350 nm, 25 and 325 nm,30 and 300 nm, 50 and 250 nm, 60 and 200 nm, 75 and 190 nm, 80 and 180nm, 100 and 200 nm, 200 and 300 nm, and 150 and 250 nm. The diameter ofan LNP may be determined by any method known in the art, for example,dynamic light scattering, transmission electron microscopy (TEM) orscanning electron microscopy (SEM). In some embodiments, an LNP has adiameter between 50 and 100 nm, between 70 and 100 nm, and between 80and 100 nm. In an embodiment, an LNP has a diameter of about 90 nm. Insome embodiments, an LNP described herein has a diameter greater thanabout 30 nm. In some embodiments, an LNP has a diameter greater thanabout 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140nm, about 160 nm, about 180 nm, about 200 nm, about 225 nm, about 250nm, about 275 nm or about 300 nm. In an embodiment, an LNP has adiameter greater than about 70 nm. In an embodiment, an LNP has adiameter greater than about 90 nm. In an embodiment, an LNP has adiameter greater than about 180 nm.

In some embodiments, a plurality of LNPs described herein has an averagediameter ranging from about 40 nm to about 180 nm. In some embodiments,a plurality of LNPs described herein has an average diameter from about50 nm to about 150 nm. In some embodiments, a plurality of LNPsdescribed herein has an average diameter from about 50 nm to about 120nm.

In some embodiments, a plurality of LNPs described herein has an averagediameter from about 60 nm to about 120 nm. In some embodiments, aplurality of LNPs has an average diameter of about 40 nm, about 45 nm,about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm

In some embodiments, a nanoparticle or plurality of nanoparticlesdescribed herein has an average neutral to negative surface charge ofless than −100 mv, for example, less than −90 mv, −80 mv, −70 mv, −60mv, −50 mv, −40 mv, −30 mv, and −20 mv. In some embodiments, ananoparticle or plurality of nanoparticles has a neutral to negativesurface charge of between −100 my and 100 my, between −75 my to 0, orbetween −50 my and −10 mv.

In some embodiments, at least 5% (e.g., at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, or atleast 99%) of the nanoparticles of a plurality of nanoparticles have anaverage neutral to negative surface charge of less than −100 mv. In someembodiments, a nanoparticle or plurality of nanoparticles has an averagesurface charge of between −20 my to +20, between −10 my and +10 mv, orbetween −5 my and +5 my at pH 7.4. LNPs that are neutral in charge haveimproved pharmacokinetics and biological performance compared tocationic LNPs.

Making Lipid Nanoparticles (LNPs)

The method of making an LNP can comprise mixing a first solution with asecond solution. Mixing can be achieved using standard liquid mixingtechniques, such as propellor mixing, vortexing solutions or preferablythrough microfluidic mixing or high efficiency T-mixing. In someembodiments, the first solution comprises a lipid or a plurality oflipids and a nucleic acid, where all components are solubilized, inwater/solvent system. The solvent may be any water miscible solvent(e.g., ethanol, methanol, isopropanol, acetonitrile, dimethylformamide,dimethylsulfoxide, dioxane or tetrahydrofuran). In some embodiments, thefirst solution comprises a small percentage of water or pH bufferedwater. The first solution may comprise up to at least 60% by volume ofwater, e.g., up to at least about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% by volume ofwater. In an embodiment, the first solution comprises between about0.05% and 60% by volume of water, e.g., between about 0.05% and 50%,about 0.05% and 40%, or about 5% and 20% by volume of water.

In some embodiments, the first solution comprises a single type oflipid, for example, an ionizable lipid, a phospholipid, a sterol, or aPEG-containing lipid. In some embodiments, the first solution comprisesa plurality of lipids. In some embodiments, the plurality comprises anionizable lipid, a phospholipid, a sterol, or a PEG-containing lipid. Insome embodiments, the plurality of lipids comprise cholesterol,1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene2000 (DMG-PEG2k) orα-(3′-{[1,2-di(myristyloxy)propanoxy] carbonylamino}propyl)-ω-methoxy,polyoxyethylene (PEG2000-C-DMG), and an ionizable lipid. The pluralityof lipids may exist in any ratio. In an embodiment, the plurality oflipids comprises an ionizable lipid or sterol, a phospholipid, a sterol,a PEG-containing lipid of the above lipids or a combination thereof in aparticular ratio (e.g., a ratio described herein).

In some embodiments, the second solution is water. In some embodiments,the second solution is an aqueous buffer with a pH between 3-6 (e.g., apH of about 3, about 4, about 5, or about 6). The second solution maycomprise a load component, e.g., a nucleic acid (e.g., mRNA). The secondsolution may comprise a small percentage of water-miscible organicsolvent. The second solution may comprise up to at least 60% by volumeof at least one water miscible organic solvent, e.g., up to at leastabout 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or any percent therebetweenby volume of at least one organic solvent (e.g., a water miscibleorganic solvent). In an embodiment, the second solution comprisesbetween about 0.05% and 60% by volume of organic solvent, e.g., betweenabout 0.05% and 50%, about 0.05% and 40%, or about 5% and 20% by volumeof organic solvent (e.g., a water miscible organic solvent). The aqueousbuffer solution can be an aqueous solution of citrate buffer. In someembodiments, the aqueous buffer solution is a citrate buffer solutionwith a pH between 4-6 (e.g., a pH of about 4, about 5, or about 6). Inan embodiment, the aqueous buffer solution is a citrate buffer solutionwith a pH of about 6.

In some embodiments, the solution comprising a mixture of the first andsecond solutions comprising the LNP suspension can be diluted. In someembodiments, the pH of the solution comprising a mixture of the firstand second solutions comprising the LNP suspension can be adjusted.Dilution or adjustment of the pH of the LNP suspension can be achievedwith the addition of water, acid, base or aqueous buffer. In someembodiments, no dilution or adjustment of the pH of the LNP suspensionis carried out. In some embodiments, both dilution and adjustment of thepH of the LNP suspension is carried out.

In some embodiments, excess reagents, solvents, unencapsulated nucleicacid maybe removed from the LNP suspension by tangential flow filtration(TFF) (e.g., diafiltration). The organic solvent (e.g., ethanol) andbuffer may also be removed from the LNP suspension with TFF. In someembodiments, the LNP suspension is subjected to dialysis and not TFF. Insome embodiments, the LNP suspension is subjected to TFF and notdialysis. In some embodiments, the LNP suspension is subjected to bothdialysis and TFF.

In one aspect, the present disclosure features a method comprisingtreating a sample of LNPs comprising nucleic acid, with a fluidcomprising a detergent (e.g., Triton X-100, or anionic detergents (suchas, but not limited to, sodium dodecyl sulfate (SDS), or non-ionicdetergent, such as but not limited to β-octylglucoside, or Zwittergent3-14) for a period of time suitable to degrade the lipid layer andthereby release the encapsulated and/or entrapped nucleic acid(s). In anembodiment, the method further comprises analyzing the sample for thepresence, absence, and/or amount of the released nucleic acid(s).

LNP Comprising Ligands

Some aspects of the disclosure relate to LNP comprising a ligand (alsoreferred herein as targeting ligand) having a binding specificity for acell surface antigen, wherein the binding of the ligand to the antigeninduces the internalization of the ligand. Some embodiments relate tocompositions comprising LNP comprising a ligand as described herein.

In some embodiment, the targeting ligand is coupled to a lipidconjugate. For example, the lipid conjugate can be a hydrophilicpolymer-lipid conjugate such as, but not limited to, PEG(2000)-DSPE orPEG(2000)-DSG. Coupling can be achieved by a variety of chemistries knowin the art (for example, see Bioconjugates Techniques (Greg T.Hermanson), 3rd Edition, 2013, Elsevier). In some embodiment, thetargeting ligand is coupled to the lipid conjugate through a linker. Thelinker molecule generally contains a hydrophilic polymer chain, such asPEG-terminally linked to a lipid domain (phospholipid or sterol) andcontains a thiol-reactive functional group such as a maleimide at theterminus. The linkers comprising PEG spacers of size,phosphatidylethanolamine (PE) lipid anchors of various hydrocarbon chainlength, and terminal maleimide or iodoacetate groups are currentlycommercially available from Avanti Polar Lipids (Alabama, USA) and NOFCorporation (Japan). One such strategy commonly used is to coupleprotein to a thiol-reactive lipopolymer linker, such as1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000 (Mal-PEG-DSPE). Preferably, the protein of interest isengineered to contain one cysteine in the C-terminus to ensure asingle-point conjugation. Alternatively, F(ab)₂ or Fab′ can beenzymatically generated from IgGs and by reduction of disulfide bondswith a reducing agent such as dithiothreitol (DTT), mercaptoethylamine,(tris(2-carboxyethyl)phosphine) TCEP-HCL generate reactive cysteinethiol groups to couple to Mal-PEG-DSPE. The reaction of Mal-PEG-DSPEwith reduced cysteine takes place in aqueous buffer at pH 5.5-7.5, forexample pH 5.5, 6, 6.5, 7, 7.5, and preferably pH 6.0.

The reaction is typically complete within 4 hours. A small amount ofcysteine or mercaptoethanol is added to react with unreacted maleimidegroups and quenches the coupling reaction. Although it is not necessaryto remove unconjugated protein prior to the subsequent membraneinsertion step it is useful to purify the conjugate for the purposes ofstorage and allow more precise characterization. Due to the large sizeof the lipopolymer micelles (equivalent molecular weight 850 kDa; Nelliset al., 2005a), size exclusion chromatography (SEC) is a convenient wayto do so. Characterization of such protein-conjugates is achieved by avariety of techniques. For example, the purity is determined by SEC,molecular weight is quantitated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), protein meltingpoints by differential scanning calorimetry (DSC), isoelectric pointdetermination by capillary electrophoresis and target binding affinityby surface plasmon resonance (BIAcore) and biolayer interferometry(ForteBio).

Examples of targeting ligands may be antibodies or antibody fragmentsagainst cell surface receptors, including the Her2 receptor, epidermalgrowth factor receptor (EGFR) receptor, Ephrin A2 receptor, CLEC9Areceptor, DEC205 receptor, CLEC4A receptor, XCR1 receptor, CD141receptor, HLA-DR receptor, transferrin receptor type 1, transferrinreceptor type 2, VEGF receptor, PDGF receptor, integrin, NGF receptor,CD19, CD20, CD22, CD33, CD43, CD38, CD56, CD69, prostate specificmembrane antigen (PSMA) or a variety of other cell surface receptors, orglycoconjugates, proteoglycans, glycoproteins, and glycolipids such asthe glycoconjugate N-acetylgalactosamine (GalNAc) ligand which binds theasialoglycoprotein receptor (ASGPR), or small molecule conjugates suchas folate-PEG-DSPE which targets the folate receptor.

In one embodiment, the targeting ligand is an anti-DEC205 antibody.DEC-205 (CD205) is a type I cell surface protein expressed primarily bydendritic cells (DC). It is found on interdigitating DC in T cell areasof lymphoid tissues, bone marrow-derived DC, Langerhan's cells, and atlow levels on macrophages and T cell and is significantly up-regulatedduring the maturation of DC. Expression of DEC-205 is positivelycorrelated with that of CD8a, both being found at high levels onlymphoid DC and at low levels on myeloid DC. DEC-205 is also expressedat moderate levels by B cells and is up-regulated during the pre-B cellto B cell transition. Recombinant anti-human DEC205 antibody iscommercially available from Creative Biolabs.

In one embodiment, antigen specific targeting on LNPs is achieved bypreparing ligand-targeted LNP by co-incubation of LNP with targetingligand-lipid conjugate. The targeting ligand-lipid conjugate may beprepared prior to LNP preparation (see Nellis et al. Biotechnol Prog.2005 January-February; 21(1):205-20).

In one embodiment, LNPs are co-incubated with antibody or fragment-PEG-phospholipid micelles or other ligand-conjugate and heated at 37°C. overnight to promote antibody conjugate insertion into the LNP outermembrane (Nellis et al. Biotechnol Prog. 2005 January-February;21(1):221-32). In another aspect, insertion can be achieved by heatingat elevated temperatures for shorter time periods, for example 0.5-8 hat 37° C., or preferably 0.5-2 h at 37° C. Micellar insertion can bequenched by lowering the temperature quickly by putting the LNPs on iceafter which they can be stored in the refrigerator at 4° C. The totalamount of lipid conjugate added can be between 0.02%-2% of total lipid,or preferably 0.1%-1%, or preferably 0.1%-0.5% total lipid. Theincorporation efficiency of antibody-lipid conjugate can be measured bySDS-PAGE after LNP dissociation by SDS or other detergents by comparisonto a standard curve of the same protein (Nellis et al. Biotechnol Prog.2005 January-February; 21(1):205-20). The insertion efficiency of othertargeting ligands can be measured by ultra high performance liquidchromatography equipped with evaporative light scattering detector(UPLC-ELSD) (Gauthier et al., J Mol Sci. 2019 Nov. 12; 20(22):5669).

FIG. 6 shows the reaction of the reduced c-terminal cysteine of a Fab′antibody fragment with a maleimide terminated-poly(ethylene glycol) 2000derivatized distearoylphosphatidylethanolamine. R¹ and R² are stearicacid. The final antibody lipopolymer conjugate is an intermediate thatis subsequently inserted into the outer lipid layer of lipidicnanoparticle to make it actively targeted.

LNP targeting can also accomplished by adding certain lipids to theformulation. For example, phosphatidylserine is known to redistribute tothe external surface of the plasma membrane during apoptosis and is amolecular cue for phagocytotic cell attraction (Fadok et al. Curr Biol.2003 Aug. 19; 13(16):R655-7). Phosphatidylserine (PS) andphosphatidylglycerol (PG) are recognized by dendritic cells and caninduce uptake and activation of dendritic cells. In one embodiment, PSor PG are added to the LNP lipid formulation at a concentration betweenabout 0.1 mol % to about 20 mol %, about 0.1 mol % to about 10 mol %,about 0.1 mol % to about 5 mol %, about 0.5 mol % to about 20 mol %,about 0.5 mol % to about 10 mol %, about 0.5 mol % to about 5 mol %,about 1 mol % to about 20 mol %, about 1 mol % to about 10 mol %, orabout 1 mol % to about 5 mol %, of the total lipid composition of theLNP.

In some aspects, a method of delivering a nucleic acid to a cell isprovided, the method comprising: contacting the cell with a compositioncomprising an LNP comprising a ligand (also referred herein as targetingligand) having a binding specificity for a cell surface antigen, whereinthe binding of the ligand to the antigen induces the internalization ofthe ligand. In some embodiments, the targeting ligand can be, but is notlimited to, an internalizing antibody, or a fragment thereof, a smallmolecule conjugates or glycoconjugates. In some embodiments, the bindingof the targeting ligand to a specific cell surface antigen induces theinternalization of the LNP with the targeting ligand attached by a cellexpressing at least 100,000 or at least 1,000,000 molecules of theantigen when contacted and incubated with the cell under internalizingconditions.

Compositions

In some embodiments, a lipidic nanoparticle composition comprises lipidsand nucleic acids, the lipidic nanoparticles comprising a compoundprovided herein, combinations thereof or pharmaceutically acceptablesalts thereof.

In some embodiments, the composition further comprises a pharmaceuticalexcipient.

In some embodiments, the lipidic nanoparticles are in an aqueous medium.

In some embodiments, the nucleic acid is entrapped in the lipidicnanoparticle with a compound disclosed herein or combinations thereof,wherein the nucleic acid is either RNA or DNA. In some embodiments, thenucleic acid is mRNA. In some embodiments, the nucleic acid is siRNA. Insome embodiments, the nucleic acid is DNA.

In some embodiments, the lipidic nanoparticle comprises a membranecomprising phosphatidylcholine and a sterol. In some embodiments, thesterol is cholesterol. In some embodiments, the lipidic nanoparticlecomprises a membrane comprising phosphatidylcholine, ionizable cationiclipid (ICL). In some embodiments, the ICL have a structure of Formula I,II, III or IV, and cholesterol, wherein the membrane separates theinside of the lipidic nanoparticles from the aqueous medium. In someembodiment, the ICL have a structure as shown in Table 1 and Table 2. Insome embodiments, the phosphatidylcholine isdistearoylphosphatidylcholine (DSPC) or hydrogenated soyphosphatidylcholine (HSPC). In some embodiments, the ionizable cationiclipid to cholesterol molar ratios is from about 65:35 to 40:60. In someembodiments, the ICL to cholesterol molar ratio is from about 60:40 toabout 45:55.

In some embodiments, the phosphatidylcholine to cholesterol molar ratiois from about 1:5 to about 1:2.

In some embodiments, the membrane further comprises a polymer-conjugatedlipid.

In some embodiments, the lipidic nanoparticle comprises ICL, DSPC,cholesterol and polymer-conjugated lipid in a about 49.5:10.3:39.6:2.5molar ratio.

In some embodiments, the polymer-conjugated lipid isPEG(2000)-dimyristoylglycerol (PEG-DMG) or PEG(Mol. weight2,000)-dimyristoylphosphatidylethanolamine (PEG-DMPE).

The compositions of this disclosure may be administered by variousroutes, for example, to effect systemic delivery via intravenous,parenteral, intraperitoneal, or topical routes. The compositions may beadministered intravenously, subcutaneously, or intraperitoneally to asubject.

In some embodiments, the disclosure provides methods for in vivodelivery of nucleic acids to a subject.

In some embodiments, the composition is a liquid pharmaceuticalformulation for parenteral administration.

In some embodiments, the composition is a liquid pharmaceuticalformulation for subcutaneous, intramuscular, or intradermaladministration.

In some embodiments, the composition is in the form of a lyophilizedpowder, that is subsequently reconstituted with aqueous medium prior toadministration.

Methods of Use

Disclosed herein are compounds or pharmaceutically acceptable saltsthereof that are useful in the preparation of vaccines. In someembodiments, the vaccine is used for the prevention Mycobacteriuminfections. In some embodiments, the vaccine can be used for theprevention of tuberculosis, nontuberculous mycobacteria (NTM),nontuberculosis lung disease, leprosy, Mycobacteriumavium-intracellulare, Mycobacterium kansasii, Mycobacterium marinum,Mycobacterium ulcerans, Mycobacterium chelonae, Mycobacterium fortuitum,Mycobacterium abscessus and other infectious diseases such ascoronaviruses (COVID-19, SARS CoV2, SARS-CoV, MERS-CoV), diphtheria,ebola, flu (Influenza), hepatitis, Hib disease, HIV/AIDS, HPV (HumanPapillomavirus), malaria, measles, meningococcal disease, mumps,norovirus, plague, pneumococcal disease, polio, respiratory syncytialvirus (RSV), rotavirus, rubella (German Measles), shingles (HerpesZoster), tetanus (Lockjaw), whooping cough (Pertussis) and zika.

Provided herein are compounds, compositions and methods for thetreatment or prevention of infectious diseases, including tuberculosis.

Targeting of Dendritic Cells

Dendritic cells (DCs) are specialized antigen-presenting cells that playa central role in initiating and regulating adaptive immunity. Owing totheir potent antigen (Ag) presentation capacity and ability to generatedistinct T-cell responses, efficient and specific delivery of Ags to DCsis the cornerstone for generating Ag-specific effector and memory cellsagainst tumors or pathogens.

Dendritic cells can be generated from human blood monocytes by addinggranulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, andIFN-gamma to differentiate monocyte-derived DC in vitro. Cells inculture exhibit both dendritic and veiled morphologies, the former beingadherent, and the latter suspended. Phenotypically, they are CD1a-/dim,CD11a+, CD11b++, CD11c+, CD14dim/−, CD16a−/dim, CD18+, CD32dim/−, CD33+,CD40+, CD45R0+, CD50+, CD54+, CD64−/dim, CD68+, CD71+, CD80dim,CD86+/++, MHC class I++/, HLA-DR++/, HLA-DP+, and HLA-DQ (Geiseler etal. Dev Immunol. 1998; 6(1-2):25-39).

Alternatively, human primary blood dendritic cell lines have beendeveloped and are commercially available from Creative Biolabs.

CD8+ T cells can produce IL2, IFN-γ, and TNF, cytokines that are knownto have critical functions during Mycobacterium tuberculosis infection.Importantly, CD8+ T cells have cytolytic functions to kill Mycobacteriumtuberculosis-infected cells via granule-mediated function (via perforin,granzymes, and granulysin) or Fas-Fas ligand interaction to induceapoptosis. In humans, CD8+ T cell can produce granulysin, which can killMycobacterium tuberculosis directly. Therefore, it is anticipated thatantigen generating mRNA LNPs delivered to DC will stimulate a CD8+ Tcell response to fight against Mycobacterium tuberculosis infection.

CD8+ T cells are able to recognize M. tuberculosis specific antigens (aspeptides) presented by classical and non-classical MHC molecules.Classically restricted CD8+ T cells have been identified that recognizeantigens presented by antigen presenting cells in the context ofclassical MHC Ia (HLA-A, -B, -C) molecules. Non-classically restrictedCD8+ T cells include those CD8+ T cells that are capable of recognizingMg antigen in the context of HLA-E molecules (non-MHC 1a), glycolipidsassociated with group 1 CD1 molecules and MHC I-related molecules (MR1)such as mucosal associated invariant T cells (MAIT). Finally, γδ T cellsrepresent a separate population of CD8 (and CD4) T cells that have bothinnate and adaptive functions in response to Mycobacterium tuberculosisinfection. CD8+ T cells have been shown to play direct functions inresponse to Mycobacterium tuberculosis infection but they also playimportant roles in orchestrating many different functions in the overallhost immune response (e.g., interaction to provide optimal CD4 T cellfunction)

In one embodiment, LNPs can be added to cultured human dendritic cellsat an appropriate concentration, (e.g. 1-5 μg/mL mRNA). After some timeto allow for cellular uptake and antigen expression, human T cells(HemaCare) can be added, and the cell culture media is sampled atvarious times for INF-γ by Elisa (R&D Systems, DIF50C). Alternatively,the cells can be analyzed by flow cytometry for CD8+ marker orintracellular INFγ production (PE anti-human IFN-γ antibody, Biolegend).

In one embodiment, LNPs can be administered into a subject at a dose of0.01-5 mg/kg mRNA by any route of administration outlined above.According to some embodiments, a proportion of LNPs are taken up DCcells, while most will accumulate in the liver and spleen. The DC cellscan express the antigenic peptide, process it for MHC I presentation andtravel to the lymph node for presentation to naïve T cells inducing aneducation of memory T-cells towards the antigen.

In one embodiment, LNPs that have been modified with a targeting ligandsuch as anti-DEC205-PEG-DSPE can be administered into a subject at adose of 0.01-5 mg/kg mRNA. According to some embodiments, a higherproportion of LNPs can be taken up DC cells, allowing for increasedproduction of antigenic peptide compared to non-targeted LNP and a moreefficient vaccination against the pathogen. Additional targeting ligandsfor dendritic cells include, but are not limited to, CLEC9A, CLEC4A,XCR1, CD141, and HLD-DR. For example, assessing the CD8+ reactivity tothe in vivo produced antigen could be accomplished by measuring INFγplasma levels by species specific IFN-gamma Quantikine ELISA Kits fromR&D Systems.

In some embodiments, LNP compositions provide desirable pharmacokineticproperties such as extended plasma half-life and stable encapsulation ofmRNA. The plasma half-life can be measured as the percentage of theinjected dose (ID) remaining in blood after 6 or 24 hours followinginjection intravenously in immunocompetent mice. The stability of theencapsulation of mRNA over 24 hours in plasma can be determined bychanges in the mRNA-to-lipid ratio (mRNA/L ratio) following ivadministration in mice. In some embodiments, the percentage ofencapsulated mRNA remaining in blood is greater than 20%, preferablygreater than 30%, and most preferably greater than 40% of the injecteddose at 6 hours. The percent retained in blood after 24 h is preferablygreater than 10%, and more preferably greater than 20% of the injecteddose.

Disclosed herein are methods for preventing mycobacteria infection, suchas Mycobacterium tuberculosis, or gram positive bacteria, such asmethicillin-resistant Staphylococcus aureus (MRSA). Additionalmycobacteria and gram positive bacteria include, but are not limited to,Mycobacterium avium complex, Mycobacterium leprae, Mycobacteriumgordonae, Mycobacterium abscessus, Mycobacterium abscessus,Mycobacterium mucogenicum, streptococci, vancomycin-resistantenterococci (VRE), Staphylococcus pneumoniae, Enterococcus faecium,Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcuspyogenes, the viridans group streptococci, Listeria monocytogenes,Nocardia, and Corynebacterium.

Administration of a vaccine for inducing a second immune response mayprovide MHC class II-presented epitopes that are capable of eliciting aCD4+ helper T cell response against cells expressing antigens from whichthe MHC presented epitopes are derived. Alternatively or additionally,administration of a vaccine for inducing a second immune response mayprovide MHC class I-presented epitopes that are capable of eliciting aCD8+ T cell response against cells expressing antigens from which theMHC presented epitopes are derived. Furthermore, administration of avaccine for inducing a second immune response may provide one or moreneo-epitopes (including known neo epitopes) as well as one or moreepitopes not containing cancer specific somatic mutations but beingexpressed by cancer cells and preferably inducing an immune responseagainst cancer cells, preferably a cancer specific immune response. Inone embodiment, administration of a vaccine for inducing a second immuneresponse provides neo-epitopes that are MHC class II-presented epitopesand/or are capable of eliciting a CD4+ helper T cell response againstcells expressing antigens from which the MHC presented epitopes arederived as well as epitopes not containing cancer-specific somaticmutations that are MHC class I-presented epitopes and/or are capable ofeliciting a CD8+ T cell response against cells expressing antigens fromwhich the MHC presented epitopes are derived. In one embodiment, theepitopes do not contain cancer-specific somatic mutations.

A “cellular immune response”, a “cellular response”, a “cellularresponse against an antigen” or a similar term is meant to include acellular response directed to cells characterized by presentation of anantigen with class I or class II MHC. The cellular response relates tocells called T cells or T-lymphocytes which act as either “helper cells”or “killer cells”. The helper T cells (also termed CD4+ T cells) play acentral role by regulating the immune response and the killer cells(also termed cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLS)kill diseased cells such as cancer cells, preventing the production ofmore diseased cells. In preferred embodiments, the present disclosureinvolves the stimulation of an anti-Mycobacterium tuberculosis CTLresponse against the Mycobacterium expressing one or more expressedantigens and preferably presenting such expressed antigens with class IMHC.

An “antigen” according to the disclosure covers any substance that willelicit an immune response. In particular, an “antigen” relates to anysubstance, preferably a peptide or protein, that reacts specificallywith antibodies or T-lymphocytes (T cells). As used herein, the term“antigen” comprises any molecule which comprises at least one epitope.Preferably, an antigen in the context of the present disclosure is amolecule which, optionally after processing, induces an immune reaction,which is preferably specific for the antigen (including cells expressingthe antigen). According to the present disclosure, any suitable antigenmay be used, which is a candidate for an immune reaction, wherein theimmune reaction is preferably a cellular immune reaction. In the contextof the embodiments of the present disclosure, the antigen is preferablypresented by a cell, preferably by an antigen presenting cell whichincludes a diseased cell, in particular a cancer cell, in the context ofMHC molecules, which results in an immune reaction against the antigen.An antigen is preferably a product which corresponds to or is derivedfrom a naturally occurring antigen. Such naturally occurring antigensmay include tumor antigens.

As used herein, an “antigen peptide” relates to a portion or fragment ofan antigen which is capable of stimulating an immune response,preferably a cellular response against the antigen or cellscharacterized by expression of the antigen and preferably bypresentation of the antigen such as diseased cells, in particular cancercells. Preferably, an antigen peptide is capable of stimulating acellular response against a cell characterized by presentation of anantigen with class I MHC and preferably is capable of stimulating anantigen-responsive cytotoxic T-lymphocyte (CTL). Preferably, the antigenpeptides according to the disclosure are MHC class I and/or class IIpresented peptides or can be processed to produce MHC class I and/orclass II presented peptides. Preferably, the antigen peptides comprisean amino acid sequence substantially corresponding to the amino acidsequence of a fragment of an antigen. Preferably, said fragment of anantigen is an MHC class I and/or class II presented peptide. Preferably,an antigen peptide according to the disclosure comprises an amino acidsequence substantially corresponding to the amino acid sequence of suchfragment and is processed to produce such fragment, i.e., an MHC class Iand/or class II presented peptide derived from an antigen. If a peptideis to be presented directly, i.e., without processing, in particularwithout cleavage, it has a length which is suitable for binding to anMHC molecule, in particular a class I MHC molecule, and preferably is7-20 amino acids in length, more preferably 7-12 amino acids in length,more preferably 8-11 amino acids in length, in particular 9 or 10 aminoacids in length.

The main types of professional antigen-presenting cells are dendriticcells, which have the broadest range of antigen presentation, and areprobably the most important antigen-presenting cells, macrophages,B-cells, and certain activated epithelial cells. Dendritic cells (DCs)are leukocyte populations that present antigens captured in peripheraltissues to T cells via both MHC class II and I antigen presentationpathways. It is well known that dendritic cells are potent inducers ofimmune responses and the activation of these cells is a critical stepfor the induction of antitumoral immunity. Dendritic cells areconveniently categorized as “immature” and “mature” cells, which can beused as a simple way to discriminate between two well characterizedphenotypes.

However, this nomenclature should not be construed to exclude allpossible intermediate stages of differentiation. Immature dendriticcells are characterized as anti gen presenting cells with a highcapacity for antigen uptake and processing, which correlates with thehigh expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class IIMHC, adhesion molecules(e.g. CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86and 4-1 BB). Dendritic cell maturation is referred to as the status ofdendritic cell activation at which such antigen-presenting dendriticcells lead to T cell priming, while presentation by immature dendriticcells results in tolerance. Dendritic cell maturation is chiefly causedby biomolecules with microbial features detected by innate receptors(bacterial DNA, viral RNA, endotoxin, etc), pro-inflammatory cytokines(TNF, IL-1, IFNs), ligation of CD40 on the dendritic cell surface byCD4OL, and substances released from cells undergoing stressful celldeath. The dendritic cells can be derived by culturing bone marrow cellsin vitro with cytokines, such as granulocyte-macrophagecolony-stimulating factor (GM CSF) and tumor necrosis factor alpha.Non-professional antigen-presenting cells do not constitutively expressthe MHC class II proteins required for interaction with naive T cells;these are expressed only upon stimulation of the non-professionalantigen-presenting cells by certain cytokines such as IFNγ. “Antigenpresenting cells” can be loaded with MHC class I presented peptides bytransducing the cells with nucleic acid, preferably mRNA, encoding apeptide or polypeptide comprising the peptide to be presented, e.g. anucleic acid encoding the antigen.

In some embodiments, a pharmaceutical composition comprising a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. As used herein, a “nucleic acid” is a deoxyribonucleicacid (DNA) or ribonucleic acid (RNA), more preferably RNA, mostpreferably in vitro transcribed RNA (IVT RNA) or synthetic RNA. Nucleicacids include according to the disclosure genomic DNA, cDNA, mRNA,recombinantly produced and chemically synthesized molecules. Accordingto the disclosure, a nucleic acid may be present as a single-stranded ordouble-stranded and linear or covalently circularly closed molecule. Anucleic acid can, according to the disclosure, be isolated. The term“isolated nucleic acid” means, according to the disclosure, that thenucleic acid (i) was amplified in vitro, for example via polymerasechain reaction (PCR), (ii) was produced recombinantly by cloning, (iii)was purified, for example, by cleavage and separation by gelelectrophoresis, or (iv) was synthesized, for example, by chemicalsynthesis. A nucleic can be employed for introduction into, i.e.transfection of cells, in particular, in the form of RNA which can beprepared by in vitro transcription from a DNA template. The RNA canmoreover be modified before application by stabilizing sequences,capping, and polyadenylation.

As used herein, the term “RNA” relates to a molecule which comprisesribonucleotide residues and preferably being entirely or substantiallycomposed of ribonucleotide residues. “Ribonucleotide” relates to anucleotide with a hydroxyl group at the 2′-position of aB-D-ribofuranosyl group. The term “RNA” comprises double-stranded RNA,single-stranded RNA, isolated RNA such as partially or completelypurified RNA, essentially pure RNA, synthetic RNA, and recombinantlygenerated RNA such as modified RNA which differs from naturallyoccurring RNA by addition, deletion, substitution and/or alteration ofone or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end (s) of a RNA or internally,for example at one or more nucleotides of the RNA. Nucleotides in RNAmolecules can also comprise non-standard nucleotides, such asnon-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs or analogs of naturally-occurring RNA.

As used herein, the term “RNA” includes and preferably relates to“mRNA”. The term “mRNA” means “messenger-RNA” and relates to a“transcript” which is generated by using a DNA template and encodes apeptide or polypeptide. Typically, an mRNA comprises a 5′-UTR, a proteincoding region, and a 3′-UTR. mRNA only possesses limited half-life incells and in vitro. In the context of the present disclosure, mRNA maybe generated by in vitro transcription from a DNA template. The term“modification” in the context of the RNA used in the present disclosureincludes any modification of an RNA which is not naturally present insaid RNA. In one embodiment of the disclosure, the RNA used according tothe invention does not have uncapped 5′-triphosphates. Removal of suchuncapped 5′-triphosphates can be achieved by treating RNA with aphosphatase. The RNA according to the disclosure may have modifiedribonucleotides in order to increase its stability and/or decreasecytotoxicity. For example, in one embodiment, in the RNA used accordingto the disclosure 5-methylcytidine is substituted partially orcompletely, preferably completely, for cytidine. Alternatively oradditionally, in one embodiment, in the RNA used according to thedisclosure pseudouridine is substituted partially or completely,preferably completely, for uridine.

In one embodiment, the term “modification” relates to providing an RNAwith a 5-cap or 5′-cap analog. The term “5-cap” refers to a capstructure found on the 5′-end of an mRNA molecule and generally consistsof a guanosine nucleotide connected to the mRNA via an unusual 5′ to 5triphosphate linkage. In one embodiment, this guanosine is methylated atthe 7-position. The term “conventional 5′-cap” refers to a naturallyoccurring RNA 5′-cap, preferably to the 7-methylguanosine cap (m′G). asused herein, the term “5′-cap” includes a 5′-cap analog that resemblesthe RNA cap structure and is modified to possess the ability tostabilize RNA and/or enhance translation of RNA if attached thereto,preferably in vivo and/or in a cell.

According to the disclosure, the stability and translation efficiency ofRNA may be modified as required. For example, RNA may be stabilized andits translation increased by one or more modifications having astabilizing effects and/or increasing translation efficiency of RNA.Such modifications are described, for example, in PCT/EP2006/009448incorporated herein by reference. In order to increase expression of theRNA used according to the present disclosure, it may be modified withinthe coding region, i.e. the sequence encoding the expressed peptide orprotein, preferably without altering the sequence of the expressedpeptide or protein, so as to increase the GC content to increase mRNAstability and to perform a codon optimization and, thus, enhancetranslation in cells.

Aspects of the disclosure relate to a method of preventing a bacterialor viral infection, the method comprising administering to a subject inneed thereof an effective amount of the composition provided herein toelicit an immune response.

Aspects of the invention provide methods of vaccinating a subjectcomprising administering to the subject a single dosage of thecompositions described herein comprising a nucleic acid (e.g. mRNA)encoding an polypeptide in an effective amount to vaccinate the subject.In some embodiments, the nucleic acid is formulated within a cationiclipidic nanoparticle. In some embodiments, the lipidic nanoparticlecomposition is administered as a single injection.

Infectious diseases such as tuberculosis, HIV/AIDS, malaria, andCOVID-19 represent significant challenges to human health. Mycobacteria,for example, is a genus of bacteria responsible for tuberculosis (TB).According to the World Health Organization, worldwide, TB is one of thetop 10 causes of death and the leading cause of death from a singleinfectious agent. Despite current best efforts, there have beensignificant challenges in the development of effective vaccines for theprevention of many infectious diseases. New efforts in theidentification of individual or combinations of antigenic peptides hashelped improved the efficiency of vaccines. Nonetheless, significantopportunities remain in the engineering of adjuvants to help efficientlydeliver and present these antigenic sequences to professional antigenpresenting cells, like dendritic cells. mRNA coding for antigenicpeptides or proteins combined with ionizable cationic lipidnanoparticles represent a particularly promising strategy in thedevelopment of a vaccine.

In some embodiments, the bacterial infection is Mycobacteriumtuberculosis infection.

In some embodiments, the viral infection is a coronavirus. In someembodiments, the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2

In some embodiments, the viral infection is HIV/AIDS.

In some embodiments, the lipidic nanoparticle is administeredparenterally.

In general, administration to a patient by intradermal injection ispossible. However, injection may also be carried out intranodally into alymph node (Maloy et al. (2001), Proc Natl Acad Sci USA 98:3299-3033).The resulting cells present the complex of interest and are recognizedby autologous cytotoxic T lymphocytes which then propagate.

In some embodiments, the compositions is administered by inhalation. Insome embodiments, the composition is formulated as nasal spray, and/oraerosol Actual dosage levels of the active agents in the pharmaceuticalcompositions disclosed herein may be varied so as to obtain an amount ofthe active agent which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

“Parenteral” as used herein in the context of administration means modesof administration other than enteral and topical administration, usuallyby injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion.

The phrases “parenteral administration” and “administered parenterally”as used herein refer to modes of administration other than enteral(i.e., via the digestive tract) and topical administration, usually byinjection or infusion, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, inhalation, subcapsular, subarachnoid,respiratory mucosal, intraspinal, epidural and intrasternal injectionand infusion. Intravenous injection and infusion are often (but notexclusively) used for liposomal drug administration.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, one or more doses may beadministered over time or the dose may be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.

In some embodiments, the dose comprises between 0.01 to 5 mg/kg ofnucleic acid. In some embodiments, the dose comprises between 0.01 to 5mg/kg of mRNA. In some embodiments, the dose comprises between 0.01 to 3mg/kg of nucleic acid. In some embodiments, the dose comprises between0.01 to 3 mg/kg of mRNA. In some embodiments, the dose comprises between0.01 to 1 mg/kg of nucleic acid. In some embodiments, the dose comprisesbetween 0.01 to 1 mg/kg of mRNA. In some embodiments, the dose comprisesbetween 0.01 to 0.5 mg/kg of nucleic acid. In some embodiments, the dosecomprises between 0.01 to 0.5 mg/kg of mRNA. In some embodiments, thedose comprises between 0.01 to 1 mg/kg of mRNA. In some embodiments, thedose comprises between 0.01 to 0.1 mg/kg of nucleic acid. In someembodiments, the dose comprises between 0.01 to 0.05 mg/kg of mRNA. Insome embodiments, the dose comprises between 0.01 to 0.1 mg/kg ofnucleic acid. In some embodiments, the dose comprises between 0.01 to0.05 mg/kg of mRNA.

The dosage of the compounds and/or of their pharmaceutically acceptablesalts or the LNPs comprising the compounds and/or of theirpharmaceutically acceptable salts may vary within wide limits and shouldnaturally be adjusted, in each particular case, to the individualconditions and to the pathogenic agent to be controlled.

ADDITIONAL EMBODIMENTS

In some embodiments, the composition further comprises a targetingligand, wherein the targeting ligand is oriented to the outside of thenanoparticle. In some embodiments, the targeting ligand is an antibody.

In some embodiments, the lipidic nanoparticles are in an aqueous medium.

In some embodiments, the nucleic acid is mRNA. In some embodiments, thenucleic acid is siRNA. In some embodiments, the nucleic acid is DNA.

In some embodiments, the lipidic nanoparticle comprises a membranecomprising phosphatidylcholine and a sterol. In some embodiments, thesterol is cholesterol. In some embodiments, the lipidic nanoparticlecomprises a membrane comprising phosphatidylcholine, ionizable cationiclipid (ICL). In some embodiments, the ICL have a structure as shown inTable 1 and Table 2. In some embodiments, the phosphatidylcholine isdistearoylphosphatidylcholine (DSPC) or hydrogenated soyphosphatidylcholine (HSPC). In some embodiments, the ionizable cationiclipid to cholesterol molar ratios is from about 65:35 to 40:60. In someembodiments, the ICL to cholesterol molar ratio is from about 60:40 toabout 45:55.

In some embodiments, the phosphatidylcholine to cholesterol molar ratiois from about 1:5 to about 1:2.

In some embodiments, the membrane further comprises a polymer-conjugatedlipid.

In some embodiments, the lipidic nanoparticle comprises ICL, DSPC,cholesterol and polymer-conjugated lipid in a about 49.5:10.3:39.6:2.5molar ratio.

In some embodiments, the polymer-conjugated lipid isPEG(2000)-dimyristoylglycerol (PEG-DMG) or PEG(Mol. weight2,000)-dimyristoylphosphatidylethanolamine (PEG-DMPE).

In some embodiments the percentage of oxidative degradation products forthe ionizable lipid is less than 50% of that for a DLin-KC2-DMA orDLin-MC3-DMA control formulation.

In some embodiments, the composition is a liquid pharmaceuticalformulation for parenteral administration.

In some embodiments, the composition is a liquid pharmaceuticalformulation for subcutaneous, intramuscular, or intradermaladministration.

In some embodiments, the composition is in the form of a lyophilizedpowder, that is subsequently reconstituted with aqueous medium prior toadministration.

Other aspects of the disclosure relate to a method of preventing abacterial or viral infection, the method comprising administering to asubject in need thereof an effective amount of the composition providedherein to elicit an immune response. Some embodiments provide methods ofvaccinating a subject in need thereof, the method comprisingadministering the composition comprising a nucleic acid encoding anantigenic protein.

In some embodiments, the composition is administered subcutaneously,intramuscularly, or intradermally.

In some embodiments, the bacterial infection is Mycobacteriumtuberculosis infection. In some embodiments, the bacterial infection isa form of nontuberculosis Mycobacterium.

In some embodiments, the viral infection is a coronavirus. In someembodiments, the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2

In some embodiments, the viral infection is HIV/AIDs.

In some embodiments, the lipidic nanoparticle is administeredparenterally.

In some embodiments, the lipidic nanoparticle composition isadministered as part of a single injection.

EXAMPLES

While this disclosure has been described in relation to certainembodiments, and many details have been set forth for purposes ofillustration, it will be apparent to those skilled in the art that thisdisclosure includes additional embodiments, and that some of the detailsdescribed herein may be varied considerably without departing from thisdisclosure. This disclosure includes such additional embodiments,modifications and equivalents. In particular, this disclosure includesany combination of the features, terms, or elements of the variousillustrative components and examples.

Example 1A: Synthesis of Ionizable Lipids

Scheme 1 Synthesis of Acid Intermediates for AKG-UO-1 to AKG-UO-3 (FIG.7 )

The acid intermediates (6Z,12Z)-6,12-octadecadienoic acid and(6Z,12Z)-6,12-hexadecadienoic acid were prepared by a general synthesis,shown in

Scheme 1, involving i) an initial Witting reaction of triphenylphosphonium ylide, prepared from 5-bromo pentanol, and the correspondingaldehyde, ii) conversion of the terminal alcohol to bromide bymesylation and substitution, iii) repeating the sequence of ylidesynthesis and Witting reaction, and finally iv) periodic acid oxidationof the terminal alcohol. The resulting acid intermediates were utilizedin the synthesis of AKG-UO-1 to AKG-UO-4, vide infra.

The acid intermediate (9Z,15Z)-9,15-octadecadienoic acid used in thesynthesis of AKG-UO-5 was prepared by a general synthesis shown inScheme 2, involving i) alkylation of silyl protected 10-hydroxy-1-decynewith (5Z)-1-bromo-5-octene, ii) catalytic hydrogenation of the alkyne toa cis-alkene, iii) removal of silyl protection on the alcohol, andfinally iv) oxidation of the terminal alcohol to the desired acid.

Scheme 3 Synthesis of Acid Intermediates for AKG-BDG-01 and AKG-BDG-02(FIG. 8 )

Synthesis of two disulfide acid intermediates used in the synthesis ofAKG-BDG-1 and AKG-BDG-2 is shown in Scheme 3. A general synthesis ofacid intermediate for AKG-BDG-1 involves i) synthesis of 4-mercaptobutyric acid from 4-bromo butyric acid, ii) reaction of 4-mercaptobutyric acid with DPS resulting in 4-(2-pyridinyldisulfanyl)butanoicacid iii) catalytic hydrogenation of 3-decyn-1-ol to a cis-alkene, iv)tosylation of the primary alcohol, v) displacement of the tosyl groupusing thiourea resulting in a terminal thiol, and finally vi) couplingof the terminal thiol with 4-(2-pyridinyldisulfanyl)butanoic acidprepared in step ii above, resulting in the disulfide containing acidintermediate. Following a similar synthetic sequence starting from3-dodecyn-1-ol yielded the second acid intermediate used in thesynthesis of AKG-BDG-2.

A general synthesis of lipids AKG-UO-1, AKG-UO-4, AKG-UO-5, AKG-BDG-1and AKG-BDG-2 shown in Scheme 4 involves the following steps: i)tosylation of the primary alcohol of the commercially available chiraldioxolane ii) displacement of the tosyl group using dimethylamineresulting in a tertiary amine, iii) acid catalyzed deprotection of thediol, and finally iv) esterification of the diol with the correspondingacid intermediates synthesized according to Schemes 1-3. AKG-UO-2 isprepared following a similar synthetic sequence starting from adifferent dioxolane and a corresponding acid intermediate, as shown inScheme 5 below.

Scheme 5 Synthesis of AKG-UO-2 (FIG. 9 )

A general synthesis of trialkyl phosphate containing lipid AKG-UO-3shown in Scheme 6 involves the following steps: i) reaction of primaryalcohol of a commercially available chiral dioxolane with methyldichlorophosphite resulting in the corresponding dialkyl chlorophosphiteii) displacement of the chloride in dialkyl chlorophosphite by treatingit with 3-bromo propanol, resulting in the corresponding trialkylphosphite iii) acid catalyzed deprotection of the diol iv)esterification of the diol with the corresponding acid intermediatesynthesized according to Scheme 1, and finally v) displacement of thebromide group using dimethylamine resulting in a tertiary amine.

Scheme 6 Synthesis of AKG-UO-3 (FIG. 10 )

Alternatively, acid intermediates having two methylene groups betweendouble bond positions in the hydrocarbon chain are synthesized asdescribed in Caballeira et al., Chem. Phys. Lipids, vol. 100, p. 33-40,1999, or as described by D'yakonov et al. (D'yakonov et al., Med. Chem.Res., 2016, vol. 25, p. 30-39; D'yakonov et al., Chem. Commun. 2013,vol. 49, p 8401-8403; D'yakonov et al., 2020, Phytochem. Rev.).

Example 1B: Synthesis of Ionizable Lipids(S)-4-(dimethylamino)butane-1,2-diyl(6Z,6′Z,12Z,12′Z)-bis(octadeca-6,12-dienoate) (AKG-UO-1, O-11956)(S)-4-(diethylamino)butane-1,2-diyl(6Z,6′Z,12Z,12′Z)-bis(octadeca-6,12-dienoate) (AKG-UO-1A, O-11955)(S)-4-(dimethylamino)butane-1,2-diyl(6Z,6′Z,12Z,12′Z)-bis(hexadeca-6,12-dienoate, AKG-UO-4, O-12401)(S)-4-(diethylamino)butane-1,2-diyl(6Z,6′Z,12Z,12′Z)-bis(hexadeca-6,12-dienoate, AKG-UO-4A, O-12402)(S)-4-(dimethylamino)butane-1,2-diyl(6Z,6′Z,11Z,11′Z)-bis(octadeca-6,11-dienoate)(AKG-UO-1a)

FIG. 11A shows Scheme 7; FIG. 11B shows Scheme 8 and FIG. 11C showsScheme 9.

Experimental Procedure

Synthesis of 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran 2

To a solution of 5-bromo-1-pentanol 1 (3.6 g, 21.6 mmol) indichloromethane (100 mL) and pyridinium p-toluene sulfonate (40 mg, 0.16mmol) at 0° C. was added 3,4-dihydro-2H-pyran (6.54 mL, 71.8 mmol). Theresulting solution was stirred at room temperature for one hour thenquenched with water. The mixture was extracted with ethyl acetate (2×100mL). The combined organics were washed with brine then dried overmagnesium sulfate, filtered, and the filtrate concentrated under vacuumto give a crude oil. The crude oil was purified by chromatography onsilica using 5-10% ethyl acetate in n-hexane as eluant to give2-((5-bromopentyl)oxy)tetrahydro-2H-pyran, 2 (4.5 g, 83%) as a clearoil.

¹H NMR (300 MHz, CDCl₃): δ ppm 4.55-4.54 (d, J=4.3 Hz, 1H), 3.92-3.72(m, 2H), 3.42-3.38 (m, 3H), 1.88-1.55 (m, 3H), 1.52-1.50 (m, 10H).

Synthesis of 2-(trideca-6,12-diyn-1-yloxy)tetrahydro-2H-pyran 4

To a solution of 1,7-Octadiyne 3 (6 mL, 45.4 mmol) andhexamethylphosphoramide (16 mL, 90.8 mmol) in tetrahydrofuran (100 mL)at −78° C. was added [2.5 M n-butyllithium in n-hexane] (18 mL, 45.4mmol) dropwise. Upon completion of addition, the solution was stirred at−78° C. for one hour then warmed to −20° C. for an additional hour. Theresulting solution was cooled once again to −78° C. whereupon a solutionof 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran, 2 (5.67 g, 22.7 mmol) intetrahydrofuran (10 mL) was added. The resulting solution was allowed towarm to room temperature and stirred for 12 hours. After 12 hours, thereaction was cooled to 0° C. and quenched with water (100 mL). Thereaction mixture was then concentrated under vacuum to removetetrahydrofuran and then diluted with n-hexane. The organics were washedwith water and brine (2×100 mL). The organic layer was dried overmagnesium sulfate, filtered, and the filtrate concentrated under vacuumto give a crude oil weighing 9 g. The crude oil was purified bychromatography on silica using 5-10% ethyl acetate in n-hexane as eluantto give 2-(trideca-6,12-diyn-1-yloxy) tetrahydro-2H-pyran, 4 (4.5 g,72%) as a clear oil.

¹H NMR (300 MHz, d^(6.)DMSO): δ ppm 4.544.53 (m, 1H), 3.72-3.61 (m, 1H),3.60-3.58 (m, 1H), 3.43-3.33 (m, 1H), 3.32-3.29 (m, 1H), 2.77-2.75 (t,J=5.8 Hz, 1H), 2.16-2.13 (m, 6H), 1.55-1.41 (m, 16H).

Representative Procedure for Alkylation of Alkynes

Synthesis of 2-(hexadeca-6,12-diyn-1-yloxy) tetrahydro-2H-pyran 7

To a solution of 2-(trideca-6,12-diyn-1-yloxy) tetrahydro-2H-pyran, 4(7.14 g, 25.86 mmol) and hexamethylphosphoramide (18 mL, 103.4 mmol) intetrahydrofuran (100 mL) at −78° C. was added [2.5 M n-butyllithium inn-hexane] (41.3 mL, 103.4 mmol) dropwise. Upon completion of addition,the solution was stirred at −78° C. for one hour then warmed to −20° C.for an additional hour. The resulting solution was cooled once again to−78° C. whereupon a solution of 1-iodopropane 5 (9.9 mL, 103.4 mmol) intetrahydrofuran (20 mL) was added. The resulting solution was allowed towarm to room temperature and stirred for 12 hours. After 12 hours, thereaction was cooled to 0° C. and quenched with water (100 mL). Thereaction mixture was then concentrated under vacuum to removetetrahydrofuran and then diluted with n-hexane. The organics were washedwith water and brine (2×100 mL). The organic layer was dried overmagnesium sulfate, filtered, and the filtrate concentrated under vacuumto give a crude oil weighing 9 g. The crude oil was purified bychromatography on silica using 5% ethyl acetate in n-hexane as eluant togive 2-(hexadeca-6,12-diyn-1-yloxy)tetrahydro-2H-pyran, 7 (5.9 g, 72%)as a clear oil.

¹H NMR (300 MHz, CDCl₃): 4.57-4.55 (m, 1H), 3.86-3.74 (m, 1H), 3.73-3.71(m, 1H), 3.50-3.39 (m, 1H), 3.37-3.36 (m, 1H), 2.16-2.11 (m, 8H),1.59-1.56 (m, 2H), 1.55-1.47 (m, 16H), 0.98-0.93 (t, J=1.6 Hz, 3H).

2-(octadeca-6,12-diyn-1-yloxy)tetrahydro-2H-pyran 8

¹H NMR (300 MHz, CDCl₃): 4.57-4.55 (m, 1H), 3.85-3.74 (m, 1H), 3.73-3.70(m, 1H), 3.50-3.38 (m, 1H), 3.36-3.35 (m, 1H), 2.23-2.12 (m, 8H),1.61-1.54 (m, 2H), 1.53-1.48 (m, 16H), 1.47-1.46 (m, 4H), 0.90-0.85 (t,J=1.6 Hz, 3H).

Representative Procedure for Reduction of Alkynes to Alkenes Using “P-2Ni”

Synthesis of2-(((6Z,12Z)-hexadeca-6,12-dien-1-yl)oxy)tetrahydro-2H-pyran 9

To a solution of Sodium borohydride (0.56 g, 14.8 mmol) in ethanol (80mL) under hydrogen blanket at 0° C. was added Nickel (II) acetatetetrahydrate (3.22 g, 12.98 mmol). Upon completion of addition, thereaction was evacuated under vacuum and flushed with hydrogen. After 10minutes of stirring, ethylenediamine (3.7 mL, 65.6 mmol), and a solutionof 2-(hexadeca-6,12-diyn-1-yloxy)tetrahydro-2H-pyran, 7 (5.9 g, 18.55mmol) in ethanol (10 mL) was added. The reaction was stirred at roomtemperature under a hydrogen balloon for 4 hours. After 4 hours, thereaction mixture was evacuated of hydrogen and then flushed withnitrogen. The crude mixture was filtered over celite, and the filtrateconcentrated under vacuum to give a crude oil weighing 4 g. The crudeoil was purified by chromatography on silica using 5-10% diethyl etherin n-hexane as eluant to give2-(((6Z,12Z)-hexadeca-6,12-dien-1-yl)oxy)tetrahydro-2H-pyran, 9 (4.67 g,78% yield) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.35-5.34 (m, 4H), 4.58-4.55 (m, 1H), 3.86-3.74(m, 1H), 3.73-3.71 (m, 1H), 3.51-3.39 (m, 1H), 3.36-3.35 (m, 1H),2.03-1.98 (m, 8H), 1.57-1.39 (m, 2H), 1.38-1.36 (m, 6H), 1.35-1.32 (m,10H), 0.91-0.86 (t, J=1.6 Hz, 3H).

¹³C NMR (300 MHz, CDCl₃): 129.98, 129.85, 98.93, 77.53, 77.10, 76.68,67.72, 62.43, 30.86, 29.71, 29.70, 29.45, 29.46, 29.44, 27.20, 27.19,26.01, 25.59, 22.98, 19.78, 13.91.

2-(((6Z,12Z)-octadeca-6,12-dien-1-yl)oxy)tetrahydro-2H-pyran 10

¹H NMR (300 MHz, CDCl₃): 5.39-5.29 (m, 4H), 4.58-4.55 (m, 1H), 3.86-3.76(m, 1H), 3.74-3.68 (m, 1H), 3.51-3.41 (m, 1H), 3.39-3.36 (m, 1H),2.14-1.97 (m, 8H), 1.56-1.38 (m, 2H), 1.37-1.35 (m, 6H), 1.34-1.28 (m,14H), 0.93-0.85 (t, J=1.6 Hz, 3H).

¹³C NMR (300 MHz, CDCl₃): 130.13, 129.97, 129.84, 129.71, 98.93, 77.53,77.10, 76.68, 67.71, 62.42, 31.62, 30.86, 29.72, 29.71, 29.47, 29.46,27.27, 27.18, 26.01, 25.59, 22.67, 19.78, 14.18.

Representative Procedure for Deprotection of Tetrahydropyranyl Ether(THP)

Synthesis of (6Z,12Z)-hexadeca-6,12-dien-1-ol 11

To a solution of2-(((6Z,12Z)-hexadeca-6,12-dien-1-yl)oxy)tetrahydro-2H-pyran, 9 (4.67 g,14.5 mmol) in methanol (20 mL) was added p-Toluenesulfonic acidmonohydrate (300 mg, 1.58 mmol) at room temperature. The resultingsolution was stirred at room temperature for 3 hours then quenched withwater. The mixture was extracted with ethyl acetate (2×50 mL). Thecombined organics were washed with water then dried over magnesiumsulfate, filtered and the filtrate concentrated under vacuum to give acrude oil weighing 4 g. The crude oil was purified by chromatography onsilica using 5-10% diethyl ether in n-hexane as eluant to give(6Z,12Z)-hexadeca-6,12-dien-1-ol, 11 (2.5 g, 72%) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.34-5.33 (m, 4H), 3.65-3.61 (m, 2H), 2.02-2.00(m, 8H), 1.36-1.34 (m, 2H), 1.34-1.25 (m, 10H), 0.89-0.86 (t, J=0.82 Hz,3H).

(6Z,12Z)-octadeca-6,12-dien-1-ol 12

¹H NMR (300 MHz, CDCl₃): 5.36-5.33 (m, 4H), 3.65-3.61 (m, 2H), 2.02-2.01(m, 8H), 1.36-1.35 (m, 2H), 1.34-1.25 (m, 14H), 0.88-0.85 (t, J=0.76 Hz,3H).

Representative Procedure for Oxidation of Alcohol to Carboxylic AcidUsing Jones Reagent

Synthesis of (6Z,12Z)-hexadeca-6,12-dienoic acid 13

A mixture of (6Z,12Z)-hexadeca-6,12-dien-1-ol, 11 (2.5 g, 10.5 mmol) andJones Reagent [2M in sulfuric acid], (10.5 mL, 21 mmol) in acetone (20mL) at 0° C. was stirred for 2 hours. The mixture was quenched withwater and extracted with ethyl acetate (2×100 mL). The combined organicswere dried over magnesium sulfate, filtered and the filtrateconcentrated under vacuum to give a crude oil. The crude oil waspurified by chromatography on silica using 20% ethyl acetate in n-hexaneas eluant to give (6Z,12Z)-hexadeca-6,12-dienoic acid, 13 (1.7 g, 68%)as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.35-5.33 (m, 4H), 2.37-2.32 (t, 2H), 2.06-1.98(m, 8H), 1.64-1.39 (m, 2H), 1.37-1.32 (m, 8H), 0.91-0.87 (t, J=0.91 Hz,3H).

(6Z,12Z)-octadeca-6,12-dienoic acid 14

¹H NMR (300 MHz, CDCl₃): 5.36-5.32 (m, 4H), 2.35-2.33 (t, 2H), 2.06-2.01(m, 8H), 1.64-1.42 (m, 2H), 1.34-1.28 (m, 12H), 0.90-0.85 (t, 3H).

Synthesis of (S)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethyl4-methylbenzenesulfonate 16

To a mixture of (S)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethan-1-ol 15 (25g, 171.1 mmol) in pyridine (30 mL) at 0° C. was addedp-Toluenesulfonylchloride (35.8 g, 188.2 mmol) and DMAP (140 mg, 1.14mmol) and the reaction was stirred at room temperature overnight. Themixture was diluted with CH₂C₂ (500 mL), washed with sat. NH₄Cl, waterand Brine. The organic layer was dried over anhydrous Na₂SO₄. Thesolvent was evaporated, and the crude residue used for the next stepwithout purification. (43.8 g, 85%).

¹H NMR (300 MHz, CDCl₃): δ ppm 7.77 (d, J=8.2 Hz, 2H), 7.34 (d, J=8.1Hz, 2H), 4.15-4.01 (m, 3H), 3.65-3.47 (m, 2H), 2.43 (s, 3H), 1.82-1.62(m, 2H), 1.32 (s, 3H), 1.27 (s, 3H).

Representative Procedure for Di-Alkylamine Substitution

Synthesis of(S)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine 19

A mixture of (S)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethyl4-methylbenzenesulfonate 16 (10 g, 33.3 mmol) and dimethylamine solution17 (166 mL, 333.3 mmol) (2M in THF) was stirred at room temperature for2 days. The mixture was concentrated, and the crude residue was dilutedwith CH₂Cl₂ (500 mL), washed with sat. NaHCO₃, water and Brine. Theorganic layer was dried over anhydrous Na₂SO₄. The solvent wasevaporated, and the crude residue was purified by flash chromatography(SiO₂:CH₂Cl₂=100% to 10% of MeOH in CH₂Cl₂ with 1% NH₄OH) and colorlessoil product 19 was obtained (2.1 g, 37%).

¹H NMR (300 MHz, CDCl₃): δ ppm 4.15-4.01 (m, 2H), 3.52 (dd, J=7.4, 7.4Hz, 1H), 2.41-2.23 (m, 2H), 2.21 (s, 6H), 1.82-1.62 (m, 2H), 1.39 (s,3H), 1.33 (s, 3H).

MS (APCI⁺): 174.1 (M+1)

(S)-2-(2,2-diethyl-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine 20

¹H NMR (300 MHz, CDCl₃): δ ppm 4.15-4.01 (m, 2H), 3.48 (dd, J=7.4, 7.4Hz, 1H), 2.48-2.43 (m, 6H), 1.82-1.62 (m, 2H), 1.36 (s, 3H), 1.27 (s,3H), 0.97 (t, J=7.2 Hz, 6H).

MS (APCI⁺): 202.2 (M+1)

Representative Procedure for Ketal Hydrolysis

Synthesis of (S)-4-(dimethylamino)butane-1,2-diol hydrochloride salt 21

To a mixture of(S)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine 19 (2g, 11.54 mmol) in MeOH (10 mL) was added 1N HCl aqueous solution (17 mL,17.3 mmol) and the reaction was heated at 80° C. for 45 min. TLC(Rf=0.1, 10% MeOH in CH₂Cl₂ with 1% NH₄OH) showed the completion ofreaction. After concentration of the reaction mixture, the crude residuewas dissolved in water (5 mL) and lyophilized overnight. Sticky syrupproduct 21 was obtained (2.1 g, quant.) as HCl salt.

¹H NMR (300 MHz, D₂O): δ ppm 3.77-3.72 (m, 1H), 3.54-3.46 (m, 2H),3.29-3.22 (m, 2H), 2.85 (s, 6H), 1.92-1.79 (m, 2H).

MS (APCI⁺): 134.1 (M+1)

(S)-4-(diethylamino)butane-1,2-diol hydrochloride salt 22

¹H NMR (300 MHz, D₂O): δ ppm 3.77-3.72 (m, 1H), 3.54-3.46 (m, 2H),3.22-3.15 (m, 6H), 1.92-1.74 (m, 2H), 1.24 (t, J=7.4 Hz, 6H).

MS (APCI⁺): 162.1 (M+1)

Representative Procedure for Di-Esterification

Synthesis of (S)-4-(dimethylamino)butane-1,2-diyl(6Z,6′Z,12Z,12′Z)-bis(octadeca-6,12-dienoate) AKG-UO-1 (O-11956)

Oxalyl chloride (0.33 mL, 3.9 mmol) was added dropwise to a solution of(6Z,12Z)-octadeca-6,12-dienoic acid, 14 (0.36 g, 1.3 mmol) indichloromethane/DMF (15 mLs, 25 μL) at 0° C. and allowed reaction towarm to room temperature and stir for one hour. After one hour, thereaction was concentrated under vacuum to dryness. The residue wasre-dissolved in dichloromethane (10 mL) and added to a mixture ofN,N-Diisopropylethylamine (2.3 mL, 10 mmol), 4-Dimethylaminopyridine(317 mg, 2.6 mmol), and (S)-4-(dimethylamino)butane-1,2-diolhydrochloride, 21 (101 mg, 0.6 mmol). The resulting solution was allowedto stir for 24 hours. After 24 hours, the reaction was cooled to 0 C andquenched with water (10 mL). The reaction mixture was extracted withdichloromethane (2×100 mL) and the organics were washed with water andbrine (2×100 mL). The organic layer was dried over magnesium sulfate,filtered, and the filtrate concentrated under vacuum to give a crudeoil. The crude oil was purified by chromatography on silica using 2%methanol in dichloromethane as eluant to give(S)-4-(dimethylamino)butane-1,2-diyl(6Z,6′Z,12Z,12′Z)-bis(octadeca-6,12-dienoate), AKG-UO-1, (0.12 g, 30%)as a yellow oil.

¹H NMR (300 MHz, CDCl₃): 5.40-5.29 (m, 8H), 5.14-5.12 (m, 1H), 4.25 (dd,J=11.8, 3.3 Hz, 1H), 4.05 (dd, J=12.1, 6.3 Hz, 1H), 2.32-2.26 (m, 6H),2.20 (s, 6H), 2.06-1.99 (m, 16H), 1.78-1.70 (m, 2H), 1.65-1.58 (m, 4H),1.42-1.25 (m, 24H), 0.90-0.85 (m, 6H).

MS (APCI⁺): 658.5 (M+1)

(S)-4-(diethylamino)butane-1,2-diyl(6Z,6′Z,12Z,12′Z)-bis(octadeca-6,12-dienoate) AKG-UO-1A (O-11955)

¹H NMR (300 MHz, CDCl₃): 5.37-5.29 (m, 8H), 5.12-5.10 (m, 1H), 4.25 (dd,J=12.1, 3.6 Hz, 1H), 4.05 (dd, J=11.8, 6.3 Hz, 1H), 2.52-2.42 (m, 6H),2.29 (t, J=7.4 Hz, 4H)), 2.06-1.99 (m, 16H), 1.78-1.70 (m, 2H),1.64-1.59 (m, 4H), 1.41-1.19 (m, 24H), 0.99 (t, J=7.1 Hz, 6H), 0.96-0.87(m, 6H).

MS (APCI⁺): 686.6 (M+1)

(S)-4-(dimethylamino)butane-1,2-diyl(6Z,6′Z,12Z,12′Z)-bis(hexadeca-6,12-dienoate) AKG-UO-4 (O-12401)

¹H NMR (300 MHz, CDCl₃): 5.39-5.29 (m, 8H), 5.13-5.12 (m, 1H), 4.24 (dd,J=11.8, 3.3 Hz, 1H), 4.05 (dd, J=11.8, 6.3 Hz, 1H), 2.32-2.27 (m, 6H),2.19 (s, 6H), 2.01-1.99 (m, 16H), 1.75-1.72 (m, 2H), 1.65-1.58 (m, 4H),1.36-1.31 (m, 16H), 0.91-0.86 (m, 6H).

MS (APCI⁺): 602.5 (M+1)

Synthesis of (S)-4-(diethylamino)butane-1,2-diyl(6Z,6′Z,12Z,12′Z)-bis(hexadeca-6,12-dienoate) AKG-UO-4A (O-12402)

¹H NMR (300 MHz, CDCl₃): 5.40-5.29 (m, 8H), 5.12-5.11 (m, 1H), 4.25 (dd,J=11.8, 3.3 Hz, 1H), 4.05 (dd, J=11.8, 6.3 Hz, 1H), 2.54-2.43 (m, 6H),2.29 (t, J=7.4 Hz, 4H), 2.11-1.96 (m, 16H), 1.74-1.65 (m, 2H), 1.65-1.59(m, 4H), 1.39-1.31 (m, 16H), 0.99 (t, J=7.1 Hz, 6H), 0.91-0.89 (m, 6H).

MS (APCI⁺): 630.5 (M+1)

Synthesis of (S)-4-(dimethylamino)butane-1,2-diyl(6Z,6′Z,11Z,11′Z)-bis(octadeca-6,11-dienoate)(AKG-UO-1a) (FIG. 12)

Experimental Procedure

Synthesis of 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran 2

To a solution of 5-bromo-1-pentanol 1 (3.6 g, 21.6 mmol) indichloromethane (100 mL) and pyridinium p-toluene sulfonate (40 mg, 0.16mmol) at 0° C. was added 3,4-dihydro-2H-pyran (6.54 mL, 71.8 mmol). Theresulting solution was stirred at room temperature for one hour thenquenched with water. The mixture was extracted with ethyl acetate (2×100mL). The combined organics were washed with brine then dried overmagnesium sulfate, filtered, and the filtrate concentrated under vacuumto give a crude oil. The crude oil was purified by chromatography onsilica using 5-10% ethyl acetate in n-hexane as eluant to give2-((5-bromopentyl)oxy)tetrahydro-2H-pyran, 2 (4.5 g, 83%) as a clearoil.

¹H NMR (300 MHz, CDCl₃): δ ppm 4.55-4.54 (d, J=4.3 Hz, 1H), 3.92-3.72(m, 2H), 3.42-3.38 (m, 3H), 1.88-1.55 (m, 3H), 1.52-1.50 (m, 10H).

Synthesis of 2-(dodeca-6,11-diyn-1-yloxy)tetrahydro-2H-pyran 4a

To a solution of 1,6-heptadiyne 3a (5 g, 54.3 mmol) andhexamethylphosphoramide (19 mL, 108 mmol) in tetrahydrofuran (100 mL) at−78° C. was added [2.5 M n-butyllithium in n-hexane] (21.7 mL, 54.3mmol) dropwise. Upon completion of addition, the solution was stirred at−78° C. for one hour then warmed to −20° C. for an additional hour. Theresulting solution was cooled once again to −78° C. whereupon a solutionof 2-((5-bromopentyl)oxy)tetrahydro-2H-pyran, 2 (6.8 g, 27.1 mmol) intetrahydrofuran (10 mL) was added. The resulting solution was allowed towarm to room temperature and stirred for 12 hours. After 12 hours, thereaction was cooled to 0° C. and quenched with water (100 mL). Thereaction mixture was then concentrated under vacuum to removetetrahydrofuran and then diluted with n-hexane. The organics were washedwith water and brine (2×100 mL). The organic layer was dried overmagnesium sulfate, filtered, and the filtrate concentrated under vacuumto give a crude oil. The crude oil was purified by chromatography onsilica using 5-10% ethyl acetate in n-hexane as eluant to give2-(dodeca-6,11-diyn-1-yloxy)tetrahydro-2H-pyran, 4a (4.1 g, 58%) as aclear oil.

¹H NMR (300 MHz, CDCl₃): δ ppm 4.57-4.56 (m, 1H), 3.96-3.82 (m, 1H),3.77-3.69 (m, 1H), 3.50-3.41 (m, 1H), 3.39-3.34 (m, 1H), 2.29-2.25 (m,4H), 2.15-2.12 (m, 2H), 1.95-1.94 (t, J=5.8 Hz, 1H), 1.73-1.43 (m, 14H).

Synthesis of 2-(octadeca-6,11-diyn-1-yloxy)tetrahydro-2H-pyran 6a

To a solution of 2-(dodeca-6,11-diyn-1-yloxy)tetrahydro-2H-pyran, 4a(4.1 g, 15.64 mmol) and hexamethylphosphoramide (11 mL, 62.6 mmol) intetrahydrofuran (100 mL) at −78° C. was added [2.5 M n-butyllithium inn-hexane] (12.5 mL, 31.3 mmol) dropwise. Upon completion of addition,the solution was stirred at −78° C. for one hour then warmed to −20° C.for an additional hour. The resulting solution was cooled once again to−78° C. whereupon a solution of 1-iodohexane 5a (9.5 mL, 62.6 mmol) intetrahydrofuran (20 mL) was added. The resulting solution was allowed towarm to room temperature and stirred for 12 hours. After 12 hours, thereaction was cooled to 0° C. and quenched with water (100 mL). Thereaction mixture was then concentrated under vacuum to removetetrahydrofuran and then diluted with n-hexane. The organics were washedwith water and brine (2×100 mL). The organic layer was dried overmagnesium sulfate, filtered, and the filtrate concentrated under vacuumto give a crude oil. The crude oil was purified by chromatography onsilica using 5% ethyl acetate in n-hexane as eluant to give2-(octadeca-6,11-diyn-1-yloxy)tetrahydro-2H-pyran, 6a (3.1 g, 57%) as aclear oil.

¹H NMR (300 MHz, CDCl₃): 4.58-4.55 (m, 1H), 3.86-3.82 (m, 1H), 3.77-3.69(m, 1H), 3.51-3.47 (m, 1H), 3.41-3.34 (m, 1H), 2.26-2.21 (m, 6H),2.14-2.12 (m, 6H), 1.66-1.26 (m, 18H), 0.93-0.85 (t, J=6.5 Hz, 3H).

Synthesis of2-(((6Z,11Z)-octadeca-6,11-dien-1-yl)oxy)tetrahydro-2H-pyran 7a

To a solution of Sodium borohydride (0.27 g, 14.8 mmol) in ethanol (50mL) under hydrogen blanket at 0° C. was added Nickel (II) acetatetetrahydrate (1.55 g, 6.25 mmol). Upon completion of addition, thereaction was evacuated under vacuum and flushed with hydrogen. After 10minutes of stirring, ethylenediamine (1.8 mL, 26.8 mmol), and a solutionof 2-(octadeca-6,11-diyn-1-yloxy)tetrahydro-2H-pyran, 6a (3.1 g, 8.93mmol) in ethanol (10 mL) was added. The reaction was stirred at roomtemperature under a hydrogen balloon for 4 hours. After 4 hours, thereaction mixture was evacuated of hydrogen and then flushed withnitrogen. The crude mixture was filtered over celite, and the filtrateconcentrated under vacuum to give a crude oil weighing 4 g. The crudeoil was purified by chromatography on silica using 5-10% diethyl etherin n-hexane as eluant to give2-(((6Z,11Z)-octadeca-6,11-dien-1-yl)oxy)tetrahydro-2H-pyran, 7a (2.86g, 92% yield) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.4-5.34 (m, 4H), 4.58-4.55 (m, 1H), 3.86-3.82(m, 1H), 3.74-3.68 (m, 1H), 3.51-3.49 (m, 1H), 3.41-3.36 (m, 1H),2.06-1.99 (m, 6H), 1.83-1.67 (m, 2H), 1.59-1.51 (m, 6H), 1.48-1.32 (m,16H), 0.92-0.85 (t, J=6.6 Hz, 3H).

Synthesis of (6Z,11Z)-octadeca-6,11-dien-1-ol 8a

Procedure previously described.

¹H NMR (300 MHz, CDCl₃): 5.37-5.33 (m, 4H), 3.65-3.61 (m, 1H), 2.06-1.99(m, 6H), 1.56-1.41 (m, 4H), 1.38-1.27 (m, 14H), 0.88-0.85 (t, J=6.6 Hz,3H).

Synthesis of (6Z,11Z)-octadeca-6,11-dienoic acid 9a

Procedure previously described.

¹H NMR (300 MHz, CDCl₃): 5.38-5.33 (m, 4H), 2.37-2.33 (t, J=5.6 Hz, 2H),2.06-1.99 (m, 6H), 1.67-1.59 (m, 2H), 1.41-1.25 (m, 14H), 0.89-0.85 (t,J=6.6 Hz, 3H).

Synthesis of (S)-4-(dimethylamino)butane-1,2-diyl(6Z,6′Z,11Z,11′Z)-bis(octadeca-6,11-dienoate)(AKG-UO-1a)

Procedure previously described.

¹H NMR (300 MHz, CDCl₃): 5.39-5.29 (m, 8H), 5.14-5.12 (m, 1H), 4.25 (dd,J=11.8, 3.3 Hz, 1H), 4.06 (dd, J=11.8, 6.3 Hz, 1H), 2.32-2.28 (m, 6H),2.20 (s, 6H), 2.03-2.01 (m, 16H), 1.74-1.64 (m, 2H), 1.62-1.60 (m, 6H),1.38-1.27 (m, 22H), 0.89-0.85 (m, 6H).

MS (APCI⁺): 658.5 (M+1)

Example 1C. Synthesis of KC-01 Series of Ionizable Lipids Synthesis of2-((S)-2,2-di((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine(AKG-KC2-01, O-12095)3-((S)-2,2-di((6Z,12Z)-octadeca-6,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine(AKG-KC3-01, O-12096) (FIG. 13) Synthesis of(6Z,12Z)-1-bromooctadeca-6,12-diene, 2

To a solution of (6Z,12Z)-octadeca-6,12-dien-1-ol, 1 (3.6 g, 13.7 mmol)in dichloromethane (50 mL) at 0° C. was added methane sulfonyl chloride(1.26 mL, 16.4 mmol) and triethylamine (3.6 mL, 20.5 mmol). Theresulting solution was warmed to room temperature and stirred for 2hours. The mixture was quenched with water and extracted withdichloromethane (2×100 mL). The combined organics were washed with brinethen dried over magnesium sulfate then filtered. The filtrate wasconcentrated under vacuum to give a crude oil. The resulting oil wasdissolved in diethyl ether (50 mL), added to a stirring slurry ofmagnesium bromide ethyl etherate (7 g, 27.4 mmol) in diethyl ether (50mL) at 0° C. The mixture was warmed to room temperature and stirred for2 hours. The reaction mixture was quenched with water and extracted withethyl acetate (2×100 mL). The combined organics were washed with brinethen dried over magnesium sulfate then filtered. The filtrate wasconcentrated under vacuum to give a crude oil. The crude oil waspurified by chromatography on silica using 5-10% ethyl acetate inn-hexane as eluant to give (6Z,12Z)-1-bromooctadeca-6,12-diene, 3 (2.9g, 8.89 mmol, 65%) as a yellow oil.

¹H NMR (300 MHz, CDCl₃): 5.36-5.33 (m, 4H), 3.42-3.37 (t, J=7.5 Hz, 2H),2.04-1.97 (m, 8H), 1.83-1.83 (m, 2H), 1.37-1.28 (m, 14H), 0.90-0.86 (t,J=6.6 Hz, 3H).

Synthesis of (6Z,12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-ol, 3

A solution of (6Z,12Z)-1-bromooctadeca-6,12-diene, 2 (2 g, 6.08 mmol) inether (10 mL) was added to a mixture of magnesium turnings (162 mg, 6.69mmol) and iodine in ether (2 mL) under argon at room temperature. Themixture stirred at room temperature for 90 minutes (magnesium turningsconsumed) whereupon ethyl formate (0.24 mL, 3.04 mmol) was added. Afterstirring for one hour at room temperature, the reaction was quenchedwith 1N HCl solution. The mixture was extracted with ethyl acetate(2×100 mL) and the combined organics washed with water then brine. Theorganics were dried under magnesium sulfate, filtered, and the filtrateconcentrated under vacuum to give a crude oil. The resulting oil wasdissolved in ethanol (10 mL) and added to a solution of potassiumhydroxide (260 mg) in water (3 mL). After stirring for 12 hours, themixture pH was adjusted 4 with 2N HCl. The aqueous solution wasextracted with dichloromethane (2×) and combined. The organics werewashed with brine then dried under magnesium sulfate and filtered. Thefiltrate was concentrated under vacuum to give a crude oil. Purificationof the crude oil on silica using 10-30% ethyl acetate in n-hexane aseluant to give (6Z,12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-ol,3 (0.29 g, 0.55 mmol, 18%) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.36-5.32 (m, 8H), 3.57 (bs, 1H), 3.33-3.32,(m, 2H), 2.13-1.97 (m, 16H), 1.36-1.29 (m, 34H), 0.90-0.86 (t, J=6.6 Hz,6H).

Synthesis of (6Z,12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-one,4

To a mixture of(6Z,12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-ol, 3 (0.29 g,0.55 mmol) and sodium carbonate (3 mg, 0.03 mmol) in dichloromethane wasadded pyridinium chlorochromate (236 mg, 1.1 mmol) at 0° C. The mixturewas warmed to room temperature and stirred for one hour. After one hour,silica gel (1 g) was added to reaction and the mixture filtered. Thefiltrate was concentrated, and the resulted oil purified on silica using10-20% ethyl acetate in n-hexane as eluant to give(6Z,12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-one, 4 (0.12 g,0.23 mmol, 42%) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.36-5.32 (m, 8H), 3.36-3.32, (m, 1H),2.40-2.35 (t, J=6.6 Hz, 3H), 2.14-2.00 (m, 16H), 1.58-1.54 (m, 4H),1.34-1.29 (m, 28H), 0.90-0.86 (t, J=6.6 Hz, 6H).

Synthesis of2-((S)-2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol, 7

A mixture of (6Z,12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-one,4 (0.12 g, 0.23 mmol),(4S)-(+)-4-(2-hydroxyethyl)-2,2-dimethyl-1,3-dioxolane 5 (0.20 g, 1.38mmol), and pyridinium p-toluene sulfonate (9 mg) in toluene (10 mL) washeated at reflux under nitrogen positive pressure. After 12 hours, themixture was concentrated under vacuum to give a crude oil. The resultingcrude oil was purified by chromatography on silica using 20-40% ethylacetate in n-hexane as eluant to give2-((S)-2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol, 7 (0.11 g, 0.17 mmol, 77%) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.36-5.32 (m, 8H), 4.25-4.20 (m, 1H), 4.10-4.06(m, 1H), 3.82-3.77 (m, 1H), 3.54-3.49 (m, 1H), 2.23-2.19 (t, J=6.6 Hz,3H), 2.14-2.00 (m, 16H), 1.84-1.78 (m, 2H), 1.62-1.51 (m, 6H), 1.34-1.29(m, 28H), 0.90-0.86 (t, J=6.6 Hz, 6H).

Synthesis of3-((S)-2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)propan-1-ol,8

A mixture of (6Z,12Z,25Z,31Z)-heptatriaconta-6,12,25,31-tetraen-19-one,4 (0.50 g, 0.95 mmol), (S)-(3)-(2,2-Dimethyl-1,3-dioxolane-4-yl)propanol6 (0.76 g, 4.75 mmol), and pyridinium p-toluene sulfonate (36 mg) intoluene (10 mL) was heated at reflux under nitrogen positive pressure.After 12 hours, the mixture was concentrated under vacuum to give acrude oil. The resulting crude oil was purified by chromatography onsilica using 20-40% ethyl acetate in n-hexane as eluant to3-((S)-2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)propan-1-ol,8 (0.48 g, 0.76 mmol, 80%) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.34-5.29 (m, 8H), 4.06-4.02 (m, 2H), 3.67-3.47(m, 2H), 3.45-3.43 (m, 1H), 2.12-2.01 (m, 16H), 1.65-1.62 (m, 8H),1.34-1.29 (m, 32H), 0.89-0.85 (t, J=6.6 Hz, 6H).

Synthesis of2-((S)-2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine,(AKG-KC2-01, O-12095)

To a solution of2-((S)-2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol, 7 (0.49 g, 0.79 mmol) in dichloromethane (10 mL) at 0° C.was added methanesulfonyl chloride (73 μL, 0.95 mmol) and triethylamine(0.26 mL, 1.2 mmol). The solution was warmed to room temperature andstirred for an addition hour. The reaction was quenched with water andextracted with dichloromethane (2×100 mL). The organics were washed withbrine then dried over magnesium sulfate and filtered. The filtrate wasconcentrated under vacuum to give a crude oil. A solution of 2Mdimethylamine (10 mL) was added to the resulting crude oil and allowedto stir for 24 hours. The mixture was then quenched with water andextracted with dichloromethane (2×100 mL). The combined organics werewashed with brine then dried over magnesium sulfate then filtered. Thefiltrate was concentrated under vacuum to give a crude oil. The crudeoil was purified by chromatography on silica using 5-100% ethyl acetatein n-hexane as eluant to give2-((S)-2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine,(AKG-KC2-01, O-12095), (206 mg, 0.32 mmol, 41%) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.35-5.32 (m, 8H), 4.08-4.03 (m, 2H), 3.47 (t,J=6.8 Hz, 1H), 2.36-2.27 (m, 2H), 2.21 (s, 6H), 2.01-1.99 (m, 16H),1.88-1.77 (m, 2H), 1.68-1.53 (m, 6H), 1.42-1.19 (m, 34H), 0.96-0.86 (t,J=3.7 Hz, 6H).

MS(APCI) for C₄₃H₇₉NO₂: 642.6

Synthesis of3-((S)-2,2-di((6Z,12Z)-octadeca-6-12-dien-4-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine,AKG-KC3-01, O-12096)

Procedure previously described.

3-((S)-2,2-di((6Z,12Z)-octadeca-6-12-dien-4-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine,(AKG-KC3-01, O-12096), (255 mg, 0.39 mmol, 51%) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.39-5.32 (m, 8H), 4.06-4.02 (m, 2H), 3.48-3.44(m, 1H), 2.35-2.30 (m, 2H), 2.25 (s, 6H), 2.01-1.98 (m, 16H), 1.70-1.51(m, 12H), 1.35-1.25 (m, 32H), 0.90-0.85 (t, J=6.6 Hz, 6H).

MS(APCI) for C₄₄H₈₁NO₂: 656.6

Synthesis of2-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine(AKG-KC2-OA, O-11880)2-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine(AKG-KC2-PA, O-11879)3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine(AKG-KC3-OA, O-11957) (FIG. 14)

Experimental Procedure (Refer to Previously Described Synthesis ofAKG-KC2-01)

Synthesis of (Z)-1-bromooctadec-9-ene 3

Procedure previously described.

(Z)-1-bromooctadec-9-ene, (6.4 g, 19.33 mmol) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.36-5.32 (m, 2H), 3.41 (t, J=7.5 Hz, 2H),2.01-1.99 (m, 4H), 1.87-1.82 (m, 2H), 1.44-1.26 (m, 22H), 0.87 (t, J=6.6Hz, 3H).

(Z)-16-bromohexadec-7-ene 4

¹H NMR (300 MHz, CDCl₃): 5.36-5.32 (m, 2H), 3.42 (t, J=7.5 Hz, 2H),2.01-1.99 (m, 4H), 1.87-1.82 (m, 2H), 1.44-1.26 (m, 18H), 0.89 (t, J=6.6Hz, 3H).

Synthesis of (9Z,28Z)-heptatriaconta-9,28-dien-19-ol 5

Procedure previously described.

(9Z,28Z)-heptatriaconta-9,28-dien-19-ol (1.2 g, 2.25 mmol, 47%) as asolid.

¹H NMR (300 MHz, CDCl₃): 5.36-5.29 (m, 4H), 3.57 (bs, 1H), 2.01-1.97 (m,8H), 1.42-1.26 (m, 53H), 0.89 (t, J=6.6 Hz, 6H).

(7Z,26Z)-tritriaconta-7,26-dien-17-ol 6

¹H NMR (300 MHz, CDCl₃): 5.36-5.29 (m, 4H), 3.57 (bs, 1H), 2.01-1.97 (m,8H), 1.42-1.26 (m, 45H), 0.89 (t, J=6.6 Hz, 6H).

Synthesis of (9Z,28Z)-heptatriaconta-9,28-dien-19-one 7

Procedure previously described.

(9Z,28Z)-heptatriaconta-9,28-dien-19-one (0.89 g, 1.67 mmol, 74%) as aclear oil.

¹H NMR (300 MHz, CDCl₃): 5.36-5.29 (m, 4H), 2.03-1.98 (m, 8H), 1.42-1.26(m, 52H), 0.90-0.89 (t, J=6.6 Hz, 6H).

(7Z,26Z)-tritriaconta-7,26-dien-17-one 8

¹H NMR (300 MHz, CDCl₃): 5.36-5.29 (m, 4H), 2.03-1.98 (m, 8H), 1.42-1.26(m, 44H), 0.90-0.89 (t, J=6.6 Hz, 6H).

Synthesis of 2-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol 9

Procedure previously described.

2-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl) ethan-1-ol (0.39g, 0.63 mmol, 74%) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.36-5.28 (m, 4H), 4.22-4.10 (m, 1H), 4.08-4.05(m, 1H), 3.82-3.79 (m, 2H), 3.48 (t, J=6.8 Hz, 1H), 2.24-2.21 (m, 1H),2.01-1.99 (m, 8H), 1.81-1.80 (m, 2H), 1.59-1.54 (m, 6H), 1.34-1.26 (m,45H), 0.87 (t, J=6.3 Hz, 6H).

Synthesis of2-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol, 10

Procedure previously described.

2-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)ethan-1-ol (1.02g, 1.65 mmol, 51%) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.36-5.29 (m, 4H), 4.23-4.10 (m, 1H), 4.07-4.05(m, 1H), 3.82-3.79 (m, 2H), 3.48 (t, J=6.6 Hz, 1H), 2.24-2.12 (m, 1H),2.01-1.97 (m, 8H), 1.84-1.78 (m, 2H), 1.57-1.55 (m, 8H), 1.34-1.29 (m,35H), 0.87 (t, J=6.3 Hz, 6H).

Synthesis of3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)propan-1-ol, 11

Procedure previously described.

3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl) propan-1-ol(0.41 g, 0.65 mmol, 76%) as a clear oil

¹H NMR (300 MHz, CDCl₃): 5.39-5.32 (m, 4H), 4.06-4.03 (m, 2H), 3.71-3.67(m, 2H), 3.47-3.46 (m, 1H), 2.01-1.99 (m, 10H), 1.66-1.59 (m, 4H),1.56-1.54 (m, 6H), 1.34-1.26 (m, 44H), 0.87 (t, J=6.3 Hz, 6H).

Synthesis of2-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine,(AKG-KC2-OA, O-11880)

Procedure previously described.

2-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine,(AKG-KC2-OA, O-11880), (200 mg, 0.31 mmol, 49%) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.38-5.28 (m, 4H), 4.08-4.01 (m, 2H), 3.48 (t,J=6.8 Hz, 1H), 2.39-2.24 (m, 2H), 2.21 (s, 6H), 2.01-1.97 (m, 8H),1.82-1.77 (m, 2H), 1.68-1.52 (m, 6H), 1.34-1.26 (m, 46H), 0.87 (t, J=6.3Hz, 6H).

MS(APCI) for C₄₃H₈₃NO₂: 646.7

Synthesis of2-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine,(AKG-KC2-PA, O-11879)

Procedure previously described.

2-((S)-2,2-di((Z)-hexadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethan-1-amine,(AKG-KC2-PA, O-11879), (195 mg, 0.33 mmol, 18%) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.35-5.28 (m, 4H), 4.08-4.02 (m, 2H), 3.48 (t,J=6.6 Hz, 1H), 2.38-2.27 (m, 2H), 2.20 (s, 6H), 2.01-1.99 (m, 8H),1.97-1.80 (m, 2H), 1.77-1.52 (m, 6H), 1.34-1.29 (m, 38H), 0.87 (t, J=6.3Hz, 6H).

MS(APCI) for C₃₉H₇₅NO₂: 590.6

Synthesis of3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine,(AKG-KC3-OA, O-11957)

Procedure previously described.

3-((S)-2,2-di((Z)-octadec-9-en-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylpropan-1-amine,(AKG-KC3-OA, O-11957), (160 mg, 0.24 mmol, 37%) as a clear oil.

¹H NMR (300 MHz, CDCl₃): 5.39-5.28 (m, 4H), 4.06-4.01 (m, 2H), 3.44 (t,J=6.8 Hz, 1H), 2.26 (t, J=6.8 Hz, 2H), 2.20 (s, 6H), 2.01-1.97 (m, 8H),1.82-1.77 (m, 2H), 1.60-1.43 (m, 8H), 1.34-1.26 (m, 46H), 0.87 (t, J=6.3Hz, 6H).

MS(APCI) for C₄₄H₈₅NO₂: 660.6

Example 2. Assay for In Vitro Cytotoxicity in Human Hepatocyte or CancerCells

LNPs can be tested in vitro over a series of 10 dilutions to determineIC50 in human hepatocyte/liver (HepG2; ATCC #HB8065) cells. As theseformulations are generally expected to be nontoxic, a positive controlof Lipofectamine™ 3000 (ThermoFisher #L3000015)-complexed mRNA (2 μlreagent/1 μg mRNA) is included in all studies. The mRNA used is CleanCapFLuc, EGFP, or MCherry reporter gene mRNA (5 moU; Trilink #L-7202,#L-7201, or #L-7203). Data is reported out as the full cell viabilitycurve, as well as a calculation of the actual IC50 value for eachcompound.

Adherent cells are grown to ˜80% confluency. The cells are trypsinizedby adding 0.25% trypsin-EDTA (Gibco #25200-072) and the cellssubsequently spun down, and 5 ml of growth medium (MEM media; Corning#10010 CM) added to disperse the cells. The cell density is determinedusing a hemocytometer. Growth medium (MEM media containing 10% FBS;Corning #35015 CV) is added to the cells to adjust to an appropriateconcentration of cells. Then, 200 μl of the cells (5,000 cells/well) isadded to a 96-well clear flat-bottom plate (Costar #9804) and incubatedin the plate at 37° C. in a humidified incubator with 5% CO₂ for 24 h.

Serial dilutions of LNP formulations using growth medium as solvent areprepared. These compounds are provided as sterile aqueous with aconcentration of 1 mg/ml mRNA. For making dilutions, each LNP stock waswarmed to room temperature. These were further diluted to 4× in thegrowth media to the highest mRNA concentration tested of 250 μg/ml.

LNPs are added to the wells at a series of 1:3 dilutions from theinitial 250 μg/ml concentration for each LNP by aspirating out the oldmedia and replacing it with 200 μl of the LNP containing media. Theplates were incubated at 37° C. in a humidified incubator with 5% CO₂for 72 h. At the end of the LNP incubation period, replace the media ineach well with 100 μl of 1× PrestoBlue Cell Viability Reagent(ThermoFisher Cat #A13261). Incubate the plate at 37° C. in a humidifiedincubator with 5% CO₂ 30 min to 2 h. Take readings at 30, 60, and 120min. Read fluorescence with 560 nm excitation and 590 nm emission usingSpectraMax M5 plate reader (Molecular Devices). Correct background bysubtracting the RFU of the control containing only the culture medium(background control well) from all sample readings. Calculate thepercentage of cytotoxicity using the formula below:

%  Cytotoxicity = [(RFU._(Meduim)−RFU_(Treamtment))/RFU._(Medium)] × 100%The IC50 was determined using GraphPad Prism using the followingformula:

Y = 100/(1 + 10^(⋀)((Log IC 50 − X)^(*)HillSlope)))

The cytotoxicity of Lipofectamine™ 3000 (ThermoFisher#L3000015)-complexed mRNA (2 μl reagent/1 μg mRNA) positive control cann some embodiments be 5-100-fold more toxic than compounds disclosedhere. This shows that disclosed compounds are less toxic than commercialtransfection reagents in an in vitro hepatocytotoxicity assay. In someembodiments, the compounds described herein form less toxic LNPs in vivothan commercially available transfection reagents.

Example 3. Determination of pKa of Ionizable Lipid

The pKa of an ionizable cationic lipid can be calculated several ways.For lipids this is sometimes difficult because membrane structure andneighboring lipids in the membrane can influence the dissociationproperties of the amino group, potentially giving inaccurate values. Anin-situ measurement is ideal, where the apparent pKa of the ionizablelipid is measured while the lipid is within its intended environment, inthis case as part of an LNP (Jayaraman 2012, Sabins 2018).

For each LNP formulation, amino lipid pKa values are determined bymeasuring the fluorescence of 2-(p-toluidino)-6-napthalene sulfonic acid(TNS) during titration from pH 3 to 12. TNS is an anionic molecule thatdoes not fluoresce in solution but increases fluorescence whenassociated with a positive lipid membrane, and this property has beenused in the past to probe membrane surface charge. A master buffer stockis prepared (10 mM sodium phosphate, 10 mM sodium borate, 10 mM sodiumcitrate, 150 mM sodium chloride) which are used to prepare buffers atvarious pH values for determining apparent pKa. Using 1 M sodiumhydroxide and 1 M hydrochloric acid, −20 unique buffers from the masterbuffer stock are prepared at different pH values between about 3 and 12.300 mM 6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt (TNSreagent) solubilized in dimethyl sulfoxide (DMSO) is used as a stock.LNP are prepared and purified into the desired pH buffer with a finalmRNA concentration of 0.04 mg/mL. Using a 96-well plate, preloaded withdesired buffers, mRNA containing LNP are added to so that the finalconcentration of mRNA is 0.7 μg/mL. To each well, TNS is added so thatthe DMSO concentration is 1% (v/v). After mixing, the fluorescence ofTNS in each well is measured (Ex/Em 331 nm/445 nm) and a sigmoidal bestfit analysis is applied to the fluorescence data. The pKa is determinedas the pH giving rise to half-maximal fluorescence intensity. Theapparent pKa measured for compounds 1-36 is within the pH range 6.0-7.0.

Example 4. Measurement of Cell Uptake of LNPs

Measurement of LNP cellular uptake is achieved by fluorescent imagingand/or fluorescent quantification. There are many suitable fluorescenttracers available, such as1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate(DiI), 3,3′-Dilinoleyloxacarbocyanine Perchlorate (DiO),1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindodicarbocyanine Perchlorate(DiD) and 1,1′-Dioctadecyl-3,3,3′,3′-TetramethylindotricarbocyanineIodide (DiR) (Thermo). These lipids are weakly fluorescent in water butexhibit high fluorescence when incorporated into lipid membranes such asthose present in LNPs. It is important that the lipids chosen arephotostable and have high extinction coefficients.

LNPs containing these types of lipids are visualized under a fluorescentmicroscope. In one method, LNP lipid formulation contains a fluorescentlipid tracer such as1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindodicarbocyanine-5,5′-DisulfonicAcid (DiI5-DS) at 0.1-0.5 mol % total lipid. Cells of interest are grownin a suitable cell culture dish, such as a 24-well plate (Corning). Thecells are seeded the day prior to the uptake study at 50% confluency andgrown overnight under appropriate conditions, for example 37° C., 5%CO2, 90-100% humidity. LNPs are added to cell culture medium at 0.1-100ug/mL mRNA, allowed to interact with the cells for some time (4-24 h),then the cells are washed with media three times to removenon-internalized LNPs before viewing. The cells are viewed with amicroscope with fluorescent detection capabilities. The relative extentof LNP cellular uptake is determined from the fluorescent intensitysignal from the cells, using non-treated cells as a background control.Alternatively, quantitative measurement of cellular fluorescent lipidmay be achieved by pelleting cells, solubilizing them with a detergentsuch as Triton-X100, and quantifying fluorescence by aspectrofluorometer or quantifying fluorescent lipid tracer by HPLC.

In a similar manner, the quantitation of fluorescently labeled mRNA isachieved. For example, dye-labeled enhanced green fluorescent protein(EGFP) and Firefly luciferase (FLuc) mRNAs, both transcribed withCyanine 5-UTP:5-Methoxy-UTP at a ratio of 1:3 is currently availablefrom Trilink Biotechnologies. Cyanine 5 has an excitation maximum of 650nm and an emission maximum of 670 nm. Substitution in this ratio resultsin mRNA that is easily visualized and that can still be translated incell culture. By entrapping fluorescently labeled mRNA one can visualizethe intracellular delivery of mRNA by methods described above.

LNP uptake in cells may be achieved by endogenous methods such as ApoEmediated, or through exogenous methods such as active targeting. It wasfound that the LNP systems containing ionizable cationic lipids takeadvantage of a “natural” targeting process where they adsorbapolipoprotein E (ApoE) in the blood (Cullis et al 2017) and are thenactively taken up in hepatocytes by a number of receptors that containApoE binding ligands (Williams et al 2010). By using non-overlappingfluorophores it is possible to independently track mRNA and LNPintracellular distribution and organelle accumulation kinetics. mRNAcellular expression levels may be quantified by using reporter systemssuch as EGFP, FLuc or mCherry, available from Trilink Biotechnologies.In one embodiment, EGFP mRNA is encapsulated in LNPs, and added to cellsof interest at 0.1-100 ug/mL mRNA. The cells may be washed free ofnon-internalized LNP by replacing the media after 4-24 h. At 24 h, GFPsignal is quantified by fluorescence microscopy or flow cytometry. Inthis way it is possible to differentiate between a panel of LNPformulations based upon reporter protein expression levels.

Example 5. Transfection Selectivity Index

A Transfection selectivity Index (TSI) is calculated to determine therelative transfection efficiency in mammalian cells, compared to therelative toxicity in those same cells. The selectivity index wascalculated using the formula below:

TSI = EF_(mammalian)/IC_(50, mammalian)

where EF_(mammalian) is the transfection efficiency expressed in termsof ng protein/million cells and IC_(50, mammalian) relates to the cellviability of the same formulation in terms of half maximal inhibitoryconcentration.

The LNPs using compounds described here (1-36) have a 50% higher TSIthan LNPs made using otherwise identical LNPs made with control moleculeDLin-MC3-DMA as the ICL

Example 6. An Assay for Lipid Peroxidation

The extent of oxidation can be determined using a forced degradationassay where LNP samples are treated with 3% H₂O₂ at 25° C., and sampledon days 0, 1, 3, and 5 for lipid oxidation products (Blessy et al.(2014) Journal of Pharmaceutical Analysis 4, 159-165). The oxidationreaction can be quenched by addition of 0.1 M butylated hydroxytoluene(BHT) in ethanol and stored frozen at −80° C. until measurement. Lipidoxidation products can be measured using a 2-thiobarbituric acid (TBA)reactivity assay (Gutteridge (1982) FEBS Letters 150, 454-458) to detectmalondialdehyde (MDA), an end product of lipid peroxidation or bydetection using an HPLC assay with evaporative light scatteringdetection (ELSD) or charged aerosol detection (CAD). Lipid oxidation andisomerization impurity structures can be assigned based on knownliterature precedent and are expected to be mixtures of isomers.

Generally, it is known in the art that lipids with multipleunsaturations in the acyl chain are more sensitive to oxidation (seeReis and Spickett (2012) Biochim Biophys Acta 1818, 2374-2387).

In some embodiments, the compounds provided herein have greater than30%, greater 50% greater 75%, greater 90%, and greater 95% reduction inoxidation byproducts when compared to the control LNP.

Example 7. Preparing Ligand-Targeted LNPs

Antibody ligands providing for specific uptake of LNPs into the cells ofinterest, such as, immune cells, in the form of antibody Fab′ fragmentsor single chain Fv fragments are prepared by any method known in the art(for example, as described in Drummond et al. U.S. Pat. Appl.20180271998; Zhou et al. U.S. Pat. No. 10,406,225; Marks et al. U.S.Pat. No. 8,974,792, which are incorporated herein by reference in theirentireties). To provide for conjugation of the ligands to LNPs, theligands are constructed with a C-terminal sequence having a Cysteineresidue, such as CAA, or GGSGGC. The ligands are expressed in bacterialor eukaryotic cells and isolated from the cellular mass or growth mediumusing standard methods such as protein affinity chromatography or metalchelation chromatography. To activate the thiol group of a terminalCysteine residue, the ligands are incubated in the presence of 15 mMCysteine in a 10 mM citrate buffer, pH 6.0-6.2, containing 140 mM NaCl,for 1 hour, and purified by gel-chromatography on a Sephadex G-25 orsimilar column, eluent 10 mM citrate buffer, pH 6.0-6.2, containing 140mM NaCl. The protein concentration in the purified, cysteine-activatedligand solution is determined using UV spectrophotometry at 280 nm. Theantibody ligand, at 1-10 mg/ml in the above named buffer, is mixed withthe aqueous solution of a maleimide-terminated PEG-DSPE derivative(mal-PEG(2000)-DSPE, cat. No. 880126, Avanti Polar Lipids, AL, USA, orSunbright® DSPE-020MA, NOF corporation, Japan) at the protein/lipidmolar ratio of 4:1. Mal-PEG-lipids having PEG spacer with molecularweight or 3,400 (Sunbright® DSPE-034MA) or 5,000, available from NOFCorporation (Sunbright® DSPE-050MA), can be used where longer distancebetween the LNP surface and the ligand moiety is desirable. The solutionis incubated at ambient temperature for 2 hours, adjusted to 0.5 mMCysteine to block unreacted maleimide groups, and the micellarligand-PEG-DSPE conjugate is purified by gel chromatography on UltrogelAcA 34 (if the ligand is a Fab) or Ultrogel AcA 44 (if the ligand is ascFv), eluent—144 mM NaCl buffered with 10 mM HEPES, pH 7.0-7.4. Theconjugated protein is quantified by UV spectrophotometry, and the purityis confirmed by SDS gel-electrophoresis.

Ligand is appended to the surface of LNPs by one of the followingmethods.

Method 1. Preformed LNPs (obtained as described in Hope et al. U.S. Pat.No. 10,653,780) are mixed with the micellar solution of theligand-PEG-DSPE conjugate in a HEPES-buffered saline (10 mM HEPES, 140mM NaCl, pH 7.0-7.2) to achieve the required ligand/lipid ratio in therange of 5-100 (typically 15-30) ligands per LNP particle. The mixtureis incubated with slow agitation 2 hours at 37-40° C., or overnight at2-8° C., during which time the conjugate is incorporated into the outerlipid layer of the LNPs. The ligand-conjugated LNPs are purified fromunincorporated ligand-PEG-DSPE by gel chromatography on Sepharose CL-2Bor CL-4B (hydrophilic size exclusion media with the same molecularweight cutoff can be also used); the LNP fraction appearing near thevoid volume is collected. The amount of ligand conjugated to theparticles is determined by SDS gel-electrophoresis with Coomassie Blueor fluorescent staining and concurrently run ligand standards.

Method 2. A solution of the ligand-PEG-DSPE conjugate in 10 mMNa-citrate buffer pH 4.0 containing also the nucleic acid component ofthe LNP is mixed with the ethanolic solution of the LNP lipids to thefinal ethanol concentration of 40% by volume as described by Semple etal., U.S. Pat. No. 8,021,686, incorporated herein by reference in itsentirety. Alternatively, a LNP-preparation protocol of Hope et al. U.S.Pat. No. 10,653,780 (incorporated herein by reference in its entirety)is employed. The amount of ligand-PEG-DSPE is 0.1-1 mol % of the lipid.The mixture is dialyzed against HEPES-buffered saline (10 mM HEPES, 140mM NaCl, pH 7.0) to remove ethanol. Ligand-PEG-DSPE is incorporated inthe resulting LNPs. Any residual ligand-PEG-DSPE is removed by gelchromatography using Sepharose CL-4B or CL-2B, eluent HEPES-bufferedsaline, or by buffer exchange for HEPES-buffered saline by tangentialflow filtration on a polysulfone membrane (flat or hollow fibercartridge) having 500 KD molecular weight cutoff.

Method 3. Mal-PEG-DSPE is combined with preformed LNPs in acitrate-buffered saline (10 mM Na-citrate buffer pH 6.0-6.2, 140 mMNaCl) in the amount of 0.1-1 mol % relative to the LNP lipid in the samemanner as ligand-PEG-DSPE of Method 1. LNPs with incorporatedmal-PE-DSPE are purified from unincorporated mal-PEG-DSPE by gelchromatography on Sepharose CL-4B in the same buffer, and incubated withthiol-activated antibody ligand (5-100 ligands pre LNP particle) for2-24 hours. Ligand-conjugated LNP so obtained are purified fromunconjugated ligand by Sepharose CL-4B gel chromatography usingHEPES-buffered saline pH 7.0 as eluent.

Method 4. Mal-PEG-DSPE is incorporated into the LNPs at 0.1-1 mol % ofthe LNP lipid in the same manner as ligand-PEG-DSPE according to Method2. The resulting Mal-PEG-conjugated LNPs are incubated with thethiol-activated ligand and purified as described in Method 3.

Method 5. The protocol of Method 4 is performed with the difference thatinstead of mal-PEG-DSPE a maleimide-conjugated lipid without a PEGspacer (mal-DSPE, Coatsome® FE-808MA3, NOF corporation, Japan) is addedto the lipid solution. The resulting maleimide-LNPs are conjugated tothiol-activated ligand as per Method 3.

Method 6. A low-molecular ligand (e.g., mannose) is conjugated to LNP bythe Methods 1 or 2 wherein a mannose-PEG-DSPE (Biochempeg Scientific,MA, USA, cat. No. 12169) is substituted for an antibody ligand-PEG-DSPE.

Example 8. Determining Optimal Ligand Density of the Ligand-TargetedLNPs

A panel of LNPs are prepared with the increasing ligand density in agiven range (2-200 ligands per LNP particle, or 5-100 ligands per LNPparticle) using any of the methods of Example 7. The LNPs arefluorescently labeled by incorporation of a fluorescently labeled lipidor fluorescently labeled nucleic acid as described in Example 4. Thelabeled ligand-conjugated LNPs are tested for the cell uptake accordingto Example 4, and the ligand content corresponding to the maximum of theligand-specific cell uptake of the LNPs is determined. The nucleic acidintracellular function (such as mRNA expression) can be used as an assayoutput (Example 4), in which case the presence of a lipid or nucleicacid detectable label is not necessary.

Example 9. Preparation of Lipidic Nanoparticles (LNPs)

mRNA modified with 5-methoxyuridine (5 moU) and coding for mCherry (Cat#L-7203) was obtained from Trilink Biotechnologies (San Diego, Calif.).All uridine nucleosides were substituted with N1-methyl-pseudouridine.To produce the mRNA, a synthetic gene encoding the mRNA sequence wascloned into a DNA plasmid. The synthetic gene was comprised of an RNApromoter, a 5′ untranslated region, mCherry protein coding sequence, a3′ untranslated region, and a poly(A) tail region of approximately 120As. The open reading frame sequence for the mCherry mRNA from TriLink(Cat #L-7203) corresponds to SEQ ID NO: 1:

AUGGUGAGCAAGGGCGAGGAGGACAACAUGGCCAUC AUCAAGGAGUUCAUGCGGUUCAAGGUGCACAUGGAGGGCAGCGUGAACGGCCACGAGUUCGAGAUCGAGG GCGAGGGCGAGGGCCGGCCCUACGAGGGCACCCAGACCGCCAAGCUGAAGGUGACCAAGGGCGGCCCCCU GCCCUUCGCCUGGGACAUCCUGAGCCCCCAGUUCAUGUACGGCAGCAAGGCCUACGUGAAGCACCCCGCC GACAUCCCCGACUACCUGAAGCUGAGCUUCCCCGAGGGCUUCAAGUGGGAGCGGGUGAUGAACUUCGAGG ACGGCGGCGUGGUGACCGUGACCCAGGACAGCAGCCUGCAGGACGGCGAGUUCAUCUACAAGGUGAAGCU GCGGGGCACCAACUUCCCCAGCGACGGCCCCGUGAUGCAGAAGAAGACCAUGGGCUGGGAGGCCAGCAGC GAGCGGAUGUACCCCGAGGACGGCGCCCUGAAGGGCGAGAUCAAGCAGCGGCUGAAGCUGAAGGACGGCG GCCACUACGACGCCGAGGUGAAGACCACCUACAAGGCCAAGAAGCCCGUGCAGCUGCCCGGCGCCUACAA CGUGAACAUCAAGCUGGACAUCACCAGCCACAACGAGGACUACACCAUCGUGGAGCAGUACGAGCGGGCC GAGGGCCGGCACAGCACCGGCGGCAUGGACGAGCUGUACAAGAGCGGCAACUGA 

Stock solutions of each lipid were prepared. Ionizable lipids wereweighed out in 4 mL glass vials (Thermo B7999-2) and dissolved inethanol (Sigma-Aldrich 200 proof, RNase free) to a final concentrationof 10 mM. Other lipids such as DSPC, Cholesterol and PEG-DMG wereweighed out and dissolved in ethanol to a concentration of 1 mM. DSPSwas dissolved in methanol (Sulpelco, Omnisolve) at a concentration of 1mM and briefly heated to 70° C. to complete its dissolution Lipidmixtures for each individual LNP were prepared by adding the desiredvolume of each lipid stock solution to a new vial, adding ethanol ifneeded to achieve a final volume of 1.2 mL. For example, an LNPformulation of AKG-UO-1/DSPC/DSPS/Chol/PEG-DMG (50/2.5/7.5/38.5/1.5 mol%), with an N/P of 5 contained 1500 nmol AKG-UO-1, 75 nmol DSPC, 225nmol DSPS, 1155 nmol Chol and 45 nmol PEG-DMG for every 100 μg of mRNAused. mRNA solutions were prepared by thawing frozen mRNA (mCherry mRNA,Trilink) vials and diluting mRNA in 6.25 mM sodium acetate (pH 5.0) to afinal concentration of 0.033 mg/mL. To prepare LNPs, a NanoAssemblrBenchtop microfluidic device (from Precision Nanosystems) was used. IfLNPs contained DSPS, the heating block accessory set to 70° C. was used,otherwise LNPs were mixed at room temperature. 3 mL of mRNA solution wasloaded into a 3 mL disposable syringe (BD 309656) and 1 ml of lipidmixture in a 1 ml syringe (BD309659) and placed in the NanoAssemblrheating block for 4 min prior to mixing. LNP formation was achieved bypumping the liquid streams through a disposable microfluidics cassetteat 3:1 aqueous: alcohol volume ratio at 6 mL/min mixing speed. Aftermixing, 3.6 mL of LNP mixture was collected, while the initial mixedvolume of 0.35 mL and last 0.05 mL of mix was discarded. Ethanol wasremoved by buffer exchange using SpectraPor dialysis tubing (12-14kMWCO) in PBS (Cytivia, SH30256.01) or by sequential concentration anddilution using Amicon Ultra-4 centrifugal concentrators (10k MWCO, at500 g).

LNPs were typically exchanged into PBS, pH 7.4 and then 15 mM Tris, pH7.4, 20% sucrose, concentrated to 20-50 ug/mL mRNA, sterile filtered(Thermo Nalgene 0.2 um #720-1320) prior to freezing by immersion inliquid nitrogen for 5 min and long-term storage at −20° C.

Example 10. LNP Characterization

A. mRNA Concentration and Relative Encapsulation EfficiencyDetermination by Fluorescent Binding Dye

-   -   Materials: Ribogreen reagent (Thermo #11491), 3×96-well plates        with lids, PBS, dissociation buffer (PBS with 10% DMSO and 1%        (wt/wt) Zwittergent 3-14 (Sigma-Aldrich #693017), mRNA, general        pipette tips & repeater pipette tips.    -   1. 5 mL of 2 μg/mL mRNA stock were prepared in DPBS or PBS    -   2. Diluted standards were prepared as follows in single wells in        a 96-well plate (Plate A);

Final [mRNA] ng/mL Vol. stock 2 μg/mL (μL) Vol. PBS (μL) 2000 400 0 1500300 100 1000 200 200 500 100 300 0 0 400

-   -   3. Using different wells in Plate A, samples were diluted to be        within the standard curve, you'll need one well per sample. For        example, if the approximate mRNA concentration should be ˜30        ug/mL in the sample, a 20× dilution was performed (Dilution        Factor). (20 uL sample added to 380 μL PBS in a well). No lid        was used on plate A. Samples were mixed by gentle pipetting up &        down.

Example of Plate A

A 0 500 1000 1500 2000 B C S 1 S 2 S 3 S 4 S 5 S 6 S 7 S 8 Etc. D E F GH

-   -   4. Two more plates, plates B & C were used. Using a multichannel        pipettor, 60 μL of each standard 2 were pipetted into wells each        (duplicate), and sample into 3 wells each (triplicate)

Example of Plate B and C

A 0 500 1000 1500 2000 B 0 500 1000 1500 2000 C S 1 S 2 S 3 S 4 S 5 S 6S 7 S 8 Etc. D S 1 S 2 S 3 S 4 S 5 S 6 S 7 S 8 Etc. E S 1 S 2 S 3 S 4 S5 S 6 S 7 S 8 Etc. F G H

-   -   5. The number of wells used on each plate was counted and 4 was        added to this number. For plate B, PBS was prepared with        Ribogreen diluted 1:100. For example, for 40 wells, 44 was used        as the number. 44×60 μL=2.64 mL Ribogreen solution needed, so        that would be 2.61 mL PBS with 26.4 μL Ribogreen.    -   6. For plate C, 2.61 mL Dissociation buffer and 26.4 uL        Ribogreen was pipetted.    -   7. Using a repeater pipette set for 60 μL, PBS+RiboGreen was        added to each well on plate B and 60 μL Dissociation        Buffer+Ribogreen to plate C. Both plates B and C were mixed on        an orbital mixer (120 rpm) for 1 min. Plate B was placed in the        dark for 15 min. Plate C was incubated at 37° C. in the dark for        10 min, followed by 5 min at RT.    -   8. Both plates were read one after the other, using Ex. 465, Em.        530 nm    -   9. Using the standard curve, the slope and intercept were        calculated and by extrapolation the mRNA concentrations of the        samples on plate B & C were calculated (average and std.dev)    -   10. Percent encapsulation efficiency (% EE) by [mRNA] plate        B/[mRNA] plate C×100 was calculated    -   11. Total [mRNA] by taking [mRNA] plate C× dilution factor was        calculated.

B. LNP Particle Size

-   -   1. 30 μL of LNP was mixed with 1.5 mL PBS in a polystyrene        cuvette (Sarstedt, #67.754) and analyzed for size using a        ZetaSizer Pro (Malvern) using ZS Xplorer software, version        number 1.4.0.105. The Z-average size and polydispersity index        value were recorded. Typically, size measurements of LNPs were        taken post LNP mixing, post buffer exchange and post sterile        filtering.

C. LNP Zeta Potential

-   -   1. 30 μL of LNP was mixed with 1.5 mL PBS and injected into a        disposable folded capillary cell (Malvern Nanoseries DTS1070)        and zeta potential measured on a ZetaSizer Pro at 25° C.

Example 11. Determination of Transfection Efficiency in Murine DendriticCells of LNPs Using mCherry mRNA

A. Cell Propagation, Transfection, Harvesting and Staining Protocol

-   -   1. MutuDC1940 cells (ABM) were grown according to supplier's        instructions in T75 flasks. When required, they were plated at        180,000 cells/well into 24-well plate one day prior to        transfection.    -   2. LNPs were added in triplicate to each well at 1 μg/mL in 1 mL        media and after 24 h the cells were washed once with DPBS (VWR        02-0119-1000).    -   3. 0.2 mL of DPBS (plus 5 mM EDTA, pH 7.4) was then added to        facilitate detachment.    -   4. The cells were placed at 37° C. for 3 min, until detached.    -   5. 0.5 ml DPBS added to each well and the liquid transferred to        a flow cytometry tube (Falcon 5 mL #352054)    -   6. The tube was centrifuged at 1100 rpm for 3-5 min and the        liquid poured off    -   7. 100 μL of Zombie Violet (Biolegend) (diluted 1:500 in PBS)        was added to each tube    -   8. The tubes were gently tapped to resuspend cells and placed in        the dark for 15 min at RT    -   9. To the cells 0.5 mL of (paraformaldehyde 4% in PBS:DPBS 1:1)        was added and the cells flicked gently to resuspend and put on        ice for 30 min. Another 2 ml PBS was added.    -   10. The cells were pelleted as above and resuspended in 0.5 mL        DPBS with 5% BSA and placed in the fridge until needed.

B. Cell Analysis

-   -   1. Cells suspensions were analyzed by an Attune N×T flow        cytometer using the VL1 and YL2 for live/dead and mCherry        fluorescence signals respectively. Gating analysis was performed        on FloJo software.

Example 12. Impact of Polyunsaturated Acylchain Composition onTransfection Activity of LNPs for KC2 and KC3 Series Lipids

The aim of this study was to directly compare KC2 and KC3 with ionizablecationic lipids of the same headgroups but varying in acyl chaincomposition. KC2 series lipids having a structure of dimethylaminoethylheadgroup structure were compared to the KC3 series containing adimethylaminopropyl-derivatized head group. The LNPs contained variousICLs (KC2, KC2-01, KC3 and KC3-01) as the ICL at an N/P ratio of 5 and50 mol % ICL, and a constant 1.5 mol % PEG-DMG. The cholesterol contentwas held constant at 38.5 mol % and the DSPC content was fixed at 10 mol%.

LNPs were analyzed and characterized as in Example 10. Transfectionefficiency was evaluated in murine dendritic cells as described inExample 11.

FIG. 2 is a graph showing a comparison of KC2 and KC3 polyunsaturatedICLs with a single methylene between the two olefins to KC2-01 andKC3-01 with four methylenes between the two olefins.

TABLE 3 Physicochemical properties of LNPs prepare with KC2 and KC3series lipids Ionizable Cationic Particle Polydispersity % Lipid (ICL)Size (nm) Index PDI Encapsulation ± SD KC2 93.7 0.01 68.2 ± 3.7 KC2-0170.7 0.06 88.9 ± 1.6 KC3 101.5 0.23 87.0 ± 2.3 KC3-01 91.6 0.03 88.9 ±2.7

The data show that ICLs containing olefins in the lipid tails separatedby at least two methylene groups have superior transfection efficiencycompared to their linoleic acid parent compounds, as judged by thehigher mCherry expression. KC2-01 was found to have 4.4-fold highermCherry expression than KC2 and KC3-01 had 3.4-fold high expression thanKC3.

Example 13. Impact of Ionizable Lipid Structure on TransfectionEfficiency Containing LNPs

The aim of this study was to explore the effect of different ICLs ontransfection efficiency in dendritic cells. LNPs were prepared asdescribed in Example 9, characterized for particle size and zetapotential as described in Example 10, and evaluated for transfectionefficiency in murine dendritic cells as described in Example 11. TheLNPs used the ionizable lipids in Table 4 had 50 mol % ICL, the DSPCphospholipid composition was 10 mol %, the cholesterol constant was 38.5mol % and PEG-DMG constant at 1.5 mol % with a constant N/P ratio of 5.UO-1 produced 4.2-fold higher mCherry expression than LNPs incorporatingthe O-11769 polyunsaturated lipid with a single methylene between it'stwo olefins.

TABLE 4 Physicochemical properties of diacyl ionizable cationic lipidswith varying acyl chain compositions. Particle Encap- Particle Size (nm)sulation Zeta Zeta Size Post-Freeze/ Efficiency Potential Potential ICL(nm) Thaw (%) mV, pH 5 mV, pH 7 AKG-UO1 98.0 122.6 75.4 ± 2.2 21.9 2.3AKG-UO1A 84.4 103.2 77.5 ± 0.9 28.0 −0.9 O-11769 83.0 89.6 87.5 ± 2.717.1 4.1 AKG-DM2-OA 77.6 111.5 64.9 ± 3.4 19.1 2.2 DODAP 69.8 68.2 83.4± 7.7 10.2 −0.7

FIG. 3 is a graph showing the impact of ionizable lipid acyl chaincomposition on transfection efficiency of mCherry mRNA LNPs in dendriticcells. “UT” corresponds to untreated sample (no LNP or sample was addedto the well).

Example 14. Oxidative Stability of ICLs

The aim of this studies was to compare stability of ionizable cationiclipids with conjugated olefins (such as KC2, KC3 and O-11769) and thosewith conjugated olefins (such as KC2-01, KC3-01 and UO-1) underaccelerated oxidation.

Individual lipid stocks (10 mM) were prepared in ethanol and stored at−20° C. Prior to the experiment, 5 mM suspensions (KC2, KC2-01, KC3,KC3-01, O-11769 and UO-1) were prepared by mixing 45 μl of 10 mM lipidstock in ethanol (Sigma-Aldrich, cat #459836) with 45 μl of ultra-purewater (Rx Biosciences, cat #P01-UPW02-1000). Liposomal formulationsbased on AKG-UO-1 and O-11769 ionizable cationic lipids (Table 5) wereprepared by combining lipid mixtures of desired composition in 1 mLethanol with 3 mL 6.25 mM sodium acetate, pH 5.0 at 6 mL/min on aNanoAssemblr (Precision Nanosystems). 3.6 mL of the mixture wasretained, while the initial 0.35 mL of the mixture and final 0.05 mLwere discarded. Ethanol was removed by buffer exchange into PBS, pH 7.4by concentrating each liposome preparation using an Aminon-Ultra 4centrifugal concentrator at 500 g for 10 min at 4° C. and diluting backto the original volume with PBS. This cycle was repeated multiple timesuntil the ethanol concentration was <1%. Finally, liposomes were sterilefiltered through 0.2 μm PES (Nalgene) syringe filters and size measuredby a ZetaSizer (Malvern). The AKG-UO-1 containing formulation (Lot#102021-6) had an average size of 81.8 nm and a PDI of 0.09, whereas theO-11769 containing formulation had an average size of 86.6 nm and a PDIof 0.10.

TABLE 5 LNP formulations used in the accelerated oxidation study.Estimated Lot# Composition MW mol % total lipid [mM] 102021-6 AKG-UO-1658.1 50 1.5 DSPC 790 10 Cholesterol 387 38.5 PEG₂₀₀₀-DMG 2500 1.5102021-7 O-11769 658.1 50 1.5 DSPC 790 10 Cholesterol 387 38.5PEG₂₀₀₀-DMG 2500 1.5

Aliquots of the liposomal formulations were stored at −80° C. and thawedbefore the experiment. A combined stock in water of 10% H₂0₂(Sigma-Aldrich, cat #H1009) and 1 mM of Fe(III)Cl (Sigma-Aldrich, cat#372870) was freshly prepared prior to the treatment. To make 1% finalconcentration of H₂0₂ and 100 μM Fe(III)Cl, 10 μl of 10% H₂0₂/1 mMFe(III)C1 stock was added to 90 μl of both liposomal formulations andindividual lipids The liposomes and individual lipids were incubatedwith H₂0₂/Fe(III)Cl at 37° C. and then 5 μl from each sample was takenat different time points (0, 3, 24, 48 and 72 hours) and dissolved in 90μl of MeOH for HPLC analysis. Degradation of the main lipid peak wasanalyzed using Thermo Scientific Vanquish Flex UHPLC occupied withCharged Aerosol Detector (CAD) and Thermo Scientific Accucore™ C18+UHPLC column (L=50 mm, D=2.1 mm, Particle Size=1.5 μm). The UHPLCoperating conditions are listed in Table 6.

TABLE 6 Chromatographic Conditions HPLC Instrument Thermo ScientificVanquish Flex UHPLC HPLC Column Accucore™ Vanquish™  C18 + UHPLC columnColumn Temperature 55° C. Flow Rate 0.5 mL/min Injection Volume 5 μLAbsorbance detection 210 nm CAD 10 Hz Run Time 15 min Sample Temperature21° C. Sample Solvent MeOH Mobile Phase Mobile Phase A: 5 mM ammoniumacetate in water (pH 4) Mobile Phase B: Methanol Time, Mobile MobileMobile phase program: min Phase A, % Phase B, % −0.5 15 85 0 15 85 2 1090 4 2 98 8 2 98 12 0 100 14 15 85 15 15 85

The data are presented as a percentage of the main lipid peak measuredat different time points relative to the lipid peak measured at timezero.

TABLE 7 Degradation of ICLs with two olefins separated by one (KC2, KC3,or O-11769) or more (KC2-01, KC3-01, UO-1, UO-6, and UO-7) methylenes.Lipid/ % of parent lipid peak at time 0 Formulation 24 h 48 h 72 h ICLLipid Suspensions KC2 35 ± 0.7  6 ± 1.9  0 ± 0.0 KC2-01 91 ± 0.3 87 ±0.9 83 ± 0.3 KC3 46 ± 1.8  1 ± 0.2  0 ± 0.0 KC3-01 83 ± 4.3 80 ± 0.4 74± 2.2 O-11769 31 ± 0.9  1 ± 0.2 0.3 ± 0.1 UO-1 78 ± 0.6 74 ± 0.1  65 ±2.5 UO-6 58 ± 0.8 32 ± 1.3  22 ± 1.8 UO-7 80 ± 1.7 71 ± 0.8  62 ± 1.0Liposome preparations UO-1 (102021-6) 86 ± 6.2 85 ± 4.2  73 ± 0.1O-11769 (102021-7) 27 ± 2.2  8 ± 1.4   4 ± 0.1

As shown in FIG. 4 and FIG. 5 , all ICLs with olefins that had fourmethylenes between the two olefins (KC2-01, KC3-01 and UO-1)demonstrates dramatically superior stability under accelerated oxidationwith hydrogen peroxide comparing to their counterparts with a singlemethylene separating the two olefins (KC2, KC3 and O-11769respectively). Even after 72 h treatment with hydrogen peroxide, themain peaks of KC2-01, KC3-01 and UO-1 stay above 70% relative to thestart of the treatment, while KC2, KC3 and O-11769 are fully degradedafter 48 hours of incubation with hydrogen peroxide. Two otherpolyunsaturated ICLs (UO-6 and UO-7) similarly showed good stability tooxidation, although the UO-7 with a hydroxyethyl substituent in the headgroup degraded more rapidly than then dimethylamino ICLs.

The stability of ICLs with olefins separated by more than one methylenewere studied as part of mRNA-free liposomal formulations using thestructurally related UO-1 and O-11769 ICLs. Other ionizable cationiclipids (KC2, KC2-01, KC3 and KC3-01) were excluded from this study sincetheir chromatographic peaks overleap with the peak of DSPC whichcompromises the data interpretation. The stability data of UO-1 andO-11769 based liposomal formulations are shown in FIG. 5 . UO-1formulated in liposomes has 73±0.05% of main UO-1 peak after 72 hours ofincubation in the presence of 1% hydrogen peroxide. In contrast, O-11769based liposomes show only 3.9+0.06% of the O-11769 peak after 72 hoursof the treatment.

The totality of this data suggests that in addition to the improvedtransfection efficiency, the ICLs with more than one methylene betweentheir olefins also display significantly improved stability to oxidativedegradation.

Various aspects of the present disclosure may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

While specific embodiments of the subject disclosure have beendiscussed, the above specification is illustrative and not restrictive.Many variations of the disclosure will become apparent to those skilledin the art upon review of this specification. The full scope of thedisclosure should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

All publications, patents and patent applications referenced in thisspecification are incorporated herein by reference in their entirety forall purposes to the same extent as if each individual publication,patent or patent application were specifically indicated to be soincorporated by reference.

What is claimed is:
 1. A compound selected from the group consisting ofcompounds 1-3 and compounds 5-8:


2. The compound of claim 1, wherein the compound is compound 1:


3. The compound of claim 1, wherein the compound is compound 2:


4. The compound of claim 1, wherein the compound is compound 3:


5. The compound of claim 1, wherein the compound is compound 5:


6. The compound of claim 1, wherein the compound is compound 6:


7. The compound of claim 1, wherein the compound is compound 7:


8. The compound of claim 1, wherein the compound is compound 8:


9. A pharmaceutical composition comprising a compound selected from thegroup consisting of compounds 1-3 and compounds 5-8:


10. The pharmaceutical composition of claim 9 comprising the compound 1:


11. The pharmaceutical composition of claim 9 comprising the compound 2:


12. The pharmaceutical composition of claim 9 comprising the compound 3:


13. The pharmaceutical composition of claim 9 comprising the compound 5:


14. The pharmaceutical composition of claim 9 comprising the compound 6:


15. The pharmaceutical composition of claim 9 comprising the compound 7:


16. The pharmaceutical composition of claim 9 comprising the compound 8: